An experiment was conducted to evaluate the application of dietary low-gossypol cottonseed meal (LCSM) in layers' diets. A total of 432 40-weekold Hy-line W36 laying hens were allocated to one of the six dietary treatments with 6 replicates of 12 birds each. The control group was fed a corn-soybean meal basal diet, and the 4 experimental diets consisted of a basal diet with 50, 98.3, 144.2, or 189 g/kg LCSM, respectively (correspondingly 25%, 50%, 75%, 100% dietary protein provided by soybean meal were replaced by LCSM). The sixth group was fed a basal diet supplemented with free gossypol (FG group). The feeding trial lasted for 12 weeks. The results showed that no significant difference was observed on egg production or feed conversion ratio (FCR, feed/egg, g/g) among all groups, but feed intake and egg weight were significantly decreased in the 189 g/kg LCSM group in weeks 46 to 51 (P < 0.05). A significant decrease was not observed in shell strength, shell thickness, and yolk color in all periods, but Haugh unit, albumen height, and egg white protein content were reduced in 189 g/kg LCSM group in weeks 46 to 51 (P < 0.05). No significant differences were found between the control and FG group. There was no obvious difference on plasma levels of total protein, blood urea nitrogen, uric acid, and activities of albumen, alanine aminotransferase, aspartate aminotransferase and the histopathology of liver, kidney, and oviduct in all groups. In conclusion, decreasing feed intake, egg weight, and egg quality were observed in the 189 g/kg LCSM group. However, the adverse effect was not relevant to the presence of FG in LCSM. Dietary LCSM supplementation of 98.3 g/kg was recommended with optimum FCR and without adverse effect on egg production, quality, and health of layers.