The present study was conducted to evaluate whether dietary pumpkin seed oil would affect laying performance, egg quality, egg cholesterol content and the egg yolk fatty acid composition in Brown layer hens. For that, 72 Brown layers, 28- week-old, were divided into 4 equal groups (n = 18) of similar mean weight according to the added doses (0, 10, 20 or 40 g/kg) of pumpkin seed oil into the standard ration for 8 weeks. Laying performance was assessed by recording initial and final body weights, weight gains, food intake and food conversion ratio, egg weight, egg production daily, egg characteristics, as well as the plasma glucose, cholesterol and triglyceride concentrations and the egg cholesterol content and the fatty acid composition. The dietary pumpkin seed oil inclusion has not significantly affected the growth performances, the laying performance or the egg quality even if the overall number and weight of eggs and also the thickness and the weight of shell tended to slightly decrease. Whereas the plasma concentrations of triglycerides remained similar in the 4 groups, the cholesterolemia tended to decline according to the dose added to the ration and the glycaemia was significantly depressed in supplemented hens (P < 0.05). In parallel, the egg yolk cholesterol content was significantly lowered in birds receiving the pumpkin seed oil in a dose related manner and the yolk fatty acid composition was significantly altered, showing increase of behenic acid with the highest dose of pumpkin seed oil, and marked increase of unsaturated acids, particularly of oleic and linolenic acids whatever the dose. These results show that pumpkin seed oil can be added to the ration without significantly altering the growth and laying performance in order to produce eggs enriched in unsaturated acids and made thin in cholesterol.