This study investigated the effects of different levels (0.05, 0.1, and 0.2%) of yeast culture supplementation on body weight, feed intake, feed conversion ratio, egg production, egg weight, egg quality traits, egg yolk fatty acid composition, and microbiological flora in feces. A total of 240 laying hens at 18-19 weeks of age were divided into four groups and fed a basal diet containing 2750 kcal/kg metabolizable energy and 16% crude protein for 16 weeks. The basal diet was supplemented with 0.05, 0.1, and 0.2% commercial yeast culture product obtained from Saccharomyces cerevisiae. The different levels of yeast culture supplementation to the diets did not statistically affect body weight change among the treatments. However, feed intake was lowest in the group fed 0.2% of yeast culture. The highest egg weights were obtained from the groups fed 0.1 and 0.2% yeast culture, when compared with control group. Regarding fatty acid composition, linolenic acid (C18:2 n6) was lowest in the group fed 0.2% yeast culture. However, yeast culture supplementation to the diet did not alter the microbial flora. Yeast culture (S. cerevisiae) supplementation to the diet of laying hens is beneficial for increasing feed intake and egg weight of laying hens without affecting the microbial flora in their digestive system.