The current study was conducted to evaluate the effect of clove bud on performance, egg quality, small intestinal histology and immunological responses of laying hens fed on different levels of ?6 to ?3 fatty acids. In this study, a total of 160 Loehmann brown hen layers of 43 wk of age were randomly distributed among 8 experimental of treatments with 5 replicates of 4 birds each. Dietary treatments consisted of three levels of clove bud (0, 0.2 and 0.4%) and high and low levels of n-6 to n-3 ratio (1.57 and 17.38) that fed during a 80 d feeding trial including 10 days for adaptation period and 70 days as main recording period. Performance parameters including feed intake, feed conversion ratio, egg production percentage, egg weight and egg mass were calculated in three times of d 35, 70 and total period of experiment. At 25 and 70 d of experiment, egg quality were evaluated. To investigate of immunological response, sheep red blood cells were injected in two times with 10 d interval, hens were bled 6 and 12 d after the first and second injection, respectively. Moreover, 10 d before the first SRBC injection, Newcastle disease virus were injected and then blood sera were taken 6 d after injection. In addition, differential leukocyte counts were evaluated at the end of experiment. Furthermore, at the end of experiment, two birds of each cage were exsanguinated to evaluate the relative weight as well as jejunal morphology. A reduction in dietary n-6 to n-3 ratio led to increase the relative weight of spleen (P 0.01), decrease eosinophils percentage (P 0.05), increase the secondary humoral response against SRBC (P 0.05), decrease ileal E.coli and salmonella population (P 0.01), increase goblet cells (P 0.05), increase lamb egg (P 0.01) at the end of experiment and to improve egg yolk color (P 0.05). Although inclusion of 0.2% clove bud caused a decrease in heterophil to lymphocyte ratio (P 0.01), an increase in the secondary titer against SRBC (P 0.05), a reduction in ileal microbial population (P 0.01), an improvement in intestinal health index (P 0.05) as well as a decrease in egg lambs, dietary inclusion of 0.4% clove buds resulted in an increase in the relative weight of spleen (P 0.01), a decrease in heterophil to lymphocyte ratio (P 0.01), an increase in the secondary antibody titer against SRBC and Newcastle virus disease (P 0.05), a reduction in ileal pathogen population (P 0.01), a decrease in egg lambs as well as an improvement in egg shell strength (P 0.05). Clove bud and n-6 to n-3 ratio combination had significant effects on the relative weight of heart (P 0.01), the secondary antibody titer against SRBC (P 0.01), ileal E.coli population and salmonella (P 0.01), jejunal villi length (P 0.05), lymphoid cell width (P 0.05), the number of lamb egg (P 0.05) at the first period of trial, egg weight at the second period of trial (P 0.05) and egg shell strength at the end of experiment (P 0.01). The results indicated that the best immunological responses and performance of laying hens were observed when they supplemented by fish oil and the highest levels of clove bud. However, the best histological responses attributed to birds received the lowest n-6 to n-3 ratio and 0.2% of clove bud. These results strengthen synergythic relation between polyunsaturated fatty acids of fish oil and antioxidant properties of clove bud on physiological responses. Keywords: clove bud, n-6 to n-3 ratio, laying hens, immunological responses, jejuna histology