The effects of supplementation of varying doses of Nigella sativa L. seeds in diet on feed intake, health, egg laying performance, serum and egg cholesterol contents, and population of intestinal bacteria in laying hens were investigated. One hundred 27-wk-old laying hens (Hisex Brown) were offered manually prepared diets supplemented with 0, 1.5, 3.5 or 4.5% seed powder for 10 weeks. N. sativa supplemented diet had no significant effects on feed intake, body weight, egg laying performances, and physical properties of eggs of the hens, however, significantly (P<0.05) decreased both serum triglycerides (about 70%) and egg cholesterol (about 43%) contents (up to 3.0% supplementation). Interestingly, N. sativa supplementation also significantly suppressed (about 25%) the population of harmful intestinal bacteria such as Escherichia coli. Our results suggest that N. sativa seed might have potential as an alternative to synthetic feed additives to formulate low cost and environment-friendly diet for the laying hens for low cholesterol eggs.