BACKGROUND: The objective of this study was to determine the effects of dietary yeast autolysate on performance, egg traits, egg cholesterol content, egg yolk fatty acid composition, lipid oxidation of egg yolk, some blood parameters and humoral immune response of laying hens during a 16 week period. A total of 225 Hyline Brown laying hens, 22 weeks of age, were allocated equally to one control group and four treatment groups. Yeast autolysate (Saccharomyces cerevisiae, Inte Wall) was used at levels of 1, 2, 3 and 4 g kg-1 in the diets of the first, second, third and fourth treatment groups respectively. RESULTS: Dietary treatments did not significantly affect body weight, feed intake and egg traits. Yeast autolysate supplementation increased egg production (P < 0.001) and egg weight (P < 0.001) and improved feed efficiency (P < 0.05). Yeast autolysate at levels of 2, 3 and 4 g kg-1 decreased egg yolk cholesterol level as mg g-1 yolk (P < 0.01) and blood serum levels of cholesterol and triglyceride (P < 0.05) and increased antibody titres to sheep red blood cells (P < 0.01). Total saturated fatty acids and the ratio of saturated/unsaturated fatty acids increased (P < 0.01) and total monounsaturated fatty acids (P < 0.001) decreased with yeast autolysate supplementation. CONCLUSION: Dietary yeast autolysate at levels of 2, 3 and 4 g kg-1 had beneficial effects on performance, egg cholesterol content and humoral immune response.It is concluded that 2 g kg-1 yeast autolysate will be enough to have beneficial effects in laying hens. © 2010 Society of Chemical Industry.