This study was conducted to evaluate the effect of Saccharomyces cerevisiae supplementation of a commercial layer diet on the laying performance, blood lipid profile, liver and yolk cholesterol content, and immune response during 8-week period. A total of 180 Hy-line Brown laying hens of 40 wk of age were allocated to 4 dietary treatments, where every 45 hens were divided into three replicates. The basal diet was supplemented with Saccharomyces cerevisiae at the levels of 0.0, 0.2, 0.4, and 0.6%. Results showed that the highest egg number and egg weight were observed with 0.6% Saccharomyces cerevisiae supplement. Supplementing diets with Saccharomyces cerevisiae at all levels (0.2, 0.4, and 0.6%) significantly (P˂0.05) increased egg mass, antibody production, cell-mediated immune response, and reduced feed consumption, plasma cholesterol, LDL-cholesterol levels and liver cholesterol concentration. Furthermore, hens fed diets supplemented with 0.4 and 0.6% Saccharomyces cerevisiae showed significant enhancement of feed conversion ratio, an increase of plasma HDL-cholesterol and a reduction of both yolk cholesterol concentration and Heterophil:lymphocyte (H/L) ratio. In conclusion, Saccharomyces cerevisiae could be used to enhance production performance and immune response, and control cholesterol content of plasma, yolk and liver.