The nettle Urticacannabina (U.cannabina) is not only a functional edible food for humans but also a potential alternative feed resource for poultry, providing protein, fatty acids, vitamins, and minerals. The present study was conducted to evaluate the effects of dietary U.cannabina on the production of high-quality eggs with enriched n-3 polyunsaturated fatty acids (PUFA) and lower cholesterol contents. One hundred and twenty laying hens were assigned to three groups [control, 15% alfalfa meal supplementation (TRTA), and 15% U.cannabina supplementation (TRTU)]. The results showed that the feed intake, yolk color, and shell thickness were increased (p U.cannabina group. Moreover, cholesterol contents of the yolk and serum were lower in the U.cannabina group (p U.cannabina group (p U.cannabina increased shell thickness, yolk n-3 PUFA levels, and yolk color, and reduced cholesterol contents of the yolk and serum without any negative impacts on health or laying performance.