The present study was designed to evaluate the influence of hemp seed (HS) supplementation on egg yolk cholesterol and fatty acid composition in laying hens. Sixty hens (Rhode Island Red x Fyoumi) were evenly distributed into four groups (three replicates per group) at the peak production (34 weeks). HS was included into the ration at the level of 0.0 (HS-0), 15 (HS-15), 20 (HS-20), and 25% (HS-25) and continued the supplementation for consecutively three weeks. At the end of the experiment, three eggs per replicate were randomly collected and analyzed for egg yolk fatty acids and cholesterol profile. The statistical analysis of the result revealed that supplementation of HS-25 significantly ( < 0.05) decreased egg yolk total cholesterol, myristic (C14:0), palmitic (C16:0), and stearic (C18:0). Similarly, total as well as individual monounsaturated fatty acids decreased significantly ( < 0.05) while total and individual polyunsaturated fatty acids increased significantly in the HS-25. In addition, total omega-3 and omega-6 increased significantly in the HS-25 group. From the present result, we concluded that addition of HS at the rate of 25% to the diet of laying hens augmented the cholesterol and fatty acids profile in egg yolk.