The aim of this study was to evaluate the effects of dietary non-gossypol cottonseed oil (CSO) or cottonseed meal (CSM) and their interactions on the texture properties, structure, nutritional composition, and edible safety of egg yolk. A total of 162 24-wk-old Hy-line Brown laying hens were randomly allocated into 9 diet treatments with 6 replicates of 3 hens per cage. A 3 × 3 factorial design using corn soybean meal-based diets supplemented with 0, 6, or 12% CSM and 0, 2, or 4% CSO in place of soybean meal and soybean oil, respectively, was utilized. The experiment lasted for 8 wk. Eggs obtained from the CSO groups had an egg yolk gel structure, and the hardness of egg yolk increased significantly (P < 0.001) after 4°C storaged for 2 wk; the texture properties of eggs storage at 25°C had opposite trend. There were no differences in texture properties of fresh egg yolk among the different groups (P > 0.05). The saturated fatty acid (SFA) content of egg yolk increased in a CSO dose-dependent manner, whereas opposite effects (P < 0.001) were found in the monounsaturated fatty acids (MUFA) and the omega-3/omega-6 fatty acid ratio. CSO-containing diets significantly reduced the cholesterol content (P < 0.05). No significant difference was observed among the different groups on the contents of moisture, crude protein, crude fat, phospholipid, potassium, or iron (P > 0.05) in the egg yolk. Total gossypol residues were increased with the increased amount of CSM (P < 0.05), and these changes were independent of time (P > 0.05). The total gossypol concentration in the yolk was 2.01 to 5.16 mg/kg. These results suggest that CSO has a key influence on egg yolk quality, reducing both its taste and nutritional value. Egg yolk gelation was significantly associated with the change of fatty acid composition caused by CSO and storage conditions. Free gossypol will remain in the egg yolk. Although the residue is low, the edible safety risk of eggs maybe exists.