Cottonseed meal (CSM) is commonly used in hens' diets to replace soybean meal (SBM). However, the molecular consequences of this substitution remains unclear. To investigate the impact of this substitution at the molecular level, iTRAQ combined with biochemical analysis was performed in Hy-Line W-36 hens supplemented with a mixed diet of CSM and SBM. Egg weight, albumen height, and Haugh unit were significantly reduced in the CSM100 group (100% crude protein of SBM replaced by CSM) compared with the SBM group (P<0.05). A total of 15 proteins, accounting for 75% of egg white proteins with various biological functions of egg whites, were found to be reduced. This finding may relate to the decrease of albumen quality in the CSM100 group. Oviduct magnum morphology and hormone analysis indicated that a reduced level of plasma progesterone caused reduced growth of the tubular gland and epithelial cells in the magnum, further decreasing egg white protein synthesis in the magnum. These findings help demonstrate the molecular mechanisms of a CSM diet that cause adverse effects on albumen quality, while also showing that SBM should not be totally replaced with CSM in a hen diet.