BACKGROUND: The effects of probiotic and calcium butyrate (Ca-butyrate) as feed additives on performance and immune response have been shown in laying hens. OBJECTIVES: The aim of the present study was to investigate the effects of different levels of probiotic and Ca-butyrate on performance, egg quality, blood parameters and immune response in laying hens. METHODS: Two hundred and eighty eight Hy-Line W-36 hens (62 to 70 wks of age) were used in a 2×3 factorial arrangement with three levels of probiotic (0, 0.07 and 0.01 percent) and two levels of Ca-butyrate (0 and 0.1 percent) in a completely randomized design of 6 treatments and 8 replicates. RESULTS: The results showed that feed intake, feed conversion ratio, egg production, shape index, yolk index, haugh unit, egg weight and specific gravity were not affected by probiotic and Ca-butyrate. While egg weight and egg mass were significantly (p<0.05) increased using probiotic compared with the control diet, such an effect was not observed due to Ca-butyrate addition. However, shell thickness showed significant increase (p<0.05) in response to Ca-butyrate. The interaction between different levels of probiotic and Ca-butyrate was only significant for feed conversion ratio (p<0.05). Antibody titer against sheep red blood cell (SRBC) was not affected (p>0.05) by experimental treatments. The concentration of serum albumin was significantly increased (p<0.05) by adding probiotic and Ca-butyrate to feed. Furthermore, significant increase (p<0.05) in total serum protein was pronounced in response to probiotic and Ca-butyrate. However, other serum metabolites (total calcium, triglycerides and cholesterol concentrations) were not affected by treatments. The relative percentage of heterophiles and heterophiles to lymphocytes ratio were significantly decreased (p<0.05) using probiotic. CONCLUSION: Dietary probiotic and Ca-butyrate can be considered as additives to improve immune responses in laying hens.