Background: This experiment was conducted to determine the effect of synthetic aflatoxin B1 and calcium bentonite clay as a mycotoxin binder in the laying hen diet on performance, egg quality and immune response to vaccination. Methods: A total of 144 Lohmann brown classic hens (22 weeks old) were divided into 4 groups of 6 replications with 6 hens each. This experimental design was completely randomized design and there were 4 experimental diets: (1) Basal diet (Positive control), (2) Basal diet +0.25% calcium bentonite clay, (3) Basal diet+200 ppb aflatoxin B1 (negative control) and (4) Basal diet+200 ppb aflatoxin B1 and +0.25% calcium bentonite clay. All hens were vaccinated with Newcastle (ND) and Infectious bronchitis (IB) vaccines during 24 weeks feeding trial. Result: At the end of the 24 weeks of feeding trial, dietary treatments did not influence the egg production, egg mass, feed conversion ratio (FCR) and IB titer of hens; however, aflatoxin B1 significantly (P less than 0.05) deceased final body weight and deceased the eggshell color score (P less than 0.05). Moreover, the ND titer was reduced by aflatoxin B1 at 8th and 12th weeks of trial (P=0.05 and less than 0.05; respectively). Thus, supplementation of 0.25% calcium bentonite clay in feed improved body weight, eggshell color and ND titer of laying hens similar to those fed the positive control diet.