The aim of this study was to evaluate the effect of different protein ingredients on performance, egg quality, organ health, and jejunum morphology of laying hens. A total of 216 32-wk-old Hy-Line W36 laying hens with similar egg production was randomly divided into 3 treatment groups with 6 replicates of 12 birds each. The experimental diets were isocaloric (metabolizable energy, 2 655 kcal/kg) and isonitrogenous (crude protein, 16.5%). The control group was fed a corn-soybean meal basal diet (SBM), and the other 2 experimental diets consisted of a basal diet with 195.0 g/kg low-gossypol cottonseed meal (LCSM) or 292.0 g/kg double-zero rapeseed meal (DRM). The feeding trial lasted 12 weeks. The egg production, daily feed intake, egg weight, and feed conversion ratio were not affected (P > 0.05) by diets used. The daily egg mass was decreased in the LCSM group in wk 7 to 12 (P < 0.05). Albumen height, Haugh unit, albumen weight, albumen proportion, and oviduct index were reduced in the 195.0 g/kg LCSM at wk 12 (P < 0.05), while yolk color was increased (P < 0.05). A lower yolk weight, yolk proportion, abdominal fat, dry matter of the liver, and fat content of the liver were observed in the DRM group at wk 12 (P < 0.05). Using LCSM or DRM as a sole protein ingredient in the diet reduced the ovomucin content of albumen when stored 28 d at 4°C. The villous height of the jejunum in the LCSM group was the lowest among treatments (P < 0.05). The mucosal columnar epithelial cell layer of the magnum in the LCSM group was incomplete, and there was a little albumen secretions in the lumen of the magnum. Together, our results suggest that the 195.0 g/kg LCSM diet may produce an adverse effect on daily egg mass and albumen quality. The adverse effect might be associated with the reduction of absorption ability in the jejunum and secretion capacity in the magnum.