Soil salinity is one of the growing issues in the world, which covers a large part of our country. Evaluation of salinity tolerance of medicinal plants for cultivation in saline areas is essential. This study was conducted to optimize callus production and then investigate the response of different genotypes of Nigella sativa to salt stress under in vitro culture. For this aim, the reaction of 10 different genotypes of Nigella sativa at different levels of salinity stress (84 and 250mM) was studied. Results of variance analysis showed that the variables of the hormone, genotype, and micro-sample had a significant effect on callus formation percentage and growth rate. The results of interaction between growth composition and genotypes showed that Nig2 genotype belonged to Fereidoonshahr had the maximum growth rate (0.52 mm/day), while Nig59 genotype from Syria had the lowest growth rate (0.18 mm/day) at growth composition of 3 mg/L Kin and 0.5 mg/L NAA compared with other genotypes. Also in growth composition of 3 mg /L BAP and 1 mg /L NAA Bap, genotype Nig1 belonged to Kerman province and Nig68 from Turkey showed the highest and lowest callus growth rate, respectively. The comparison of mean interaction effects between genotype and micro-sample showed that the Nig2 genotype had the highest callus growth rate (0.55 mm/day) in the leaf sample and the Nig78 genotype had the highest callus growth rate (0.35 mm/day) in hypocotyl sample. Comparison of mea interaction effects of growth composition and micro-sample showed that the highest growth rate of callus (0.39 mm/day) belonged to leaf micro-sample in growth composition of 3 mg/L BAP and 1 mg/L NAA. Thus, it gives the leaf micro-sample as the best-known micro-sample. In the second part of the study, different concentrations of sodium chloride salt (84, 250mM) and control were evaluated to examine the response of genotypes to salinity stress. The results of this study showed that with increasing salinity, sodium ion content, total phenol content, flavonoid, flavonol, anthocyanin and antioxidant activity (by DPPH method), and total thymol content increased significantly. Potassium ion content increased with salinity concentration. Nig1 and Nig2 genotypes were identified as in vitro salt-tolerant genotypes. Kywords: Callus growth rate, Antioxidant Activity, Cell cultuer, Total phenol content