This study was conducted in two experiments investigating the effects of an enzymatically hydrolyzed cottonseed protein (HCSP) as a peptide source in Holstein calves’ diet on the performance, blood metabolites, gastrointestinal development, and intestinal microbes. In the first experiment, forty-eight newborn calves were randomly assigned to one of the dietary treatments including 0, 2, 4 and 6% of HCSP (DM basis). All calves received the same amount of pasteurized whole milk, weaned on d 56 of the experiment and remained in the study until d 70. Data were analyzed using PROC MIXED in SAS (SAS Institute Inc., Cary, NC) as a randomized complete block design with linear and quadratic contrasts. Results showed that increased amount of HCSP linearly decreased the starter intake during the postweaning and overall period. In addition, when dietary HCSP increased during the overall period, ADG tended to linearly decrease. All skeletal growth parameters also linearly decreased as dietary HCSP increased at the end of the study, except for body length. Serum cortisol concentration was higher in calves supplemented with 6% of HCSP at weaning and at the end of the study indicating a stressful condition experience in these animals compared with others. Total antioxidant capacity was quadratically affected by HCSP supplementation; calves fed 2 and 4% of HCSP diets had the highest total antioxidant capacity at weaning and at end of the study. Calves supplemented with 6% HCSP had lower empty reticulo-rumen and omasum weights and rumen wall thickness compared with calves in other treatments at the end of the study. In conclusion, results of the first experiment showed that adding 2% of HCSP improved the antioxidant capacity of calves at the weaning and overall period, but did not enhance the performance and rumen development parameters. Supplementing 6% of HCSP in the starter impaired the growth responses and ruminal and intestinal development while promoting a stress condition as evidenced with greater cortisol concentration. Overall, it appears that pre-ruminant calves would not benefit from the addition of HCSP in their starter diet. The second experiment was performed with fifty-six newborn Holstein calves which were randomly assigned to one of the four dietary treatments in a 2×2 factorial arrangements with colostrum quality (good or poor) and HCSP addition to colostrum and milk (0 and 2% fresh-matter basis) from birth to 14 days. Milk feeding and weaning programs, and the duration of the experiment were the same as the first one. Our results showed that performance responses and skeletal growth parameters were not affected by colostrum quality, 2% HCSP supplementation, or their interactions, except for starter intake which increased when calves received 2% HCSP and poor quality colostrum. Calves received good quality colostrum had lower ear disposition score and those supplemented with 2% HCSP had lower nasal discharge and fecal scores, as well as fewer days with abnormal fecal score. Calves supplemented with 2% HCSP had greater ?-Hydroxybutyrate (BHBA) concentration at pre-weaning period, but tended to have lower non-esterified fatty acids (NEFA) concentrations at post-weaning period. Serum total antioxidant capacity (TAC) was greater in calves supplemented with 2% HCSP compared to un-supplemented ones and this response was more profound in those received poor quality at day 2. Serum TAC, also, tended to be greater in calves supplemented with 2% HCSP compared to un-supplemented ones during pre-weaning and post-weaning periods. Serum malondialdehyde (MDA) concentration tended to be lower in calves received good quality colostrum compared to those received poor quality colostrum at day 70. Serum cortisol concentration was lower in calves received good quality colostrum compared to those received poor quality colostrum at day 2 of the study. In addition, serum cortisol concentration tended to be lower in calves supplemented with 2% HCSP compared to un-supplemented ones at day 70. In conclusion, result of the second experiment showed that supplemented 2% HCSP improved health and metabolism of neonatal calves with more significant effects on those received poor quality colostrum. Key words: cottonseed protein hydrolysates, colostrum quality, anti-oxidant status, small peptides