To investigate the effects of different levels of cottonseed meal added to the feed on the growth, skin color, flesh color, serum biochemical indices and intestinal flora structure of channel catfish (Ictalurus punctatus), seven hundred and fifty channel catfish with an initial body mass of (49.95 ± 0.05) g were selected and randomly divided into three groups of five nets each, with 50 fish in each net cage. The base feed without cottonseed meal substitution was used as the control group, and three isonitrogenous and isolipid test feeds were formulated with 7% (group M1) and 14% (group M2) cottonseed meal replacing 25.8% and 51.7% of soybean meal in the base feed, respectively for 60 days. The experimental results showed that: Compared with the control group, cottonseed meal substituted for soybean meal had no significant effect on weight gain rate, feed coefficient and morphological indexes of channel catfish; Cottonseed meal substituted for soybean meal had no significant effect on serum levels of complement 3, complement 4, total cholesterol and triglycerides; Compared with the control group, cottonseed meal substituted for soybean meal had no significant effect on lateral skin of channel catfish, but the high cottonseed meal group (M2) significantly increased the brightness of the abdominal skin; cottonseed meal significantly increased the redness of the back muscles of the channel catfish, but there was no significant change in the brightness and yellowness values among the treatment groups. The cottonseed meal group had no significant effect on the brightness, redness and yellowness of the abdominal skin of the channel catfish. Compared with the control group, the low cottonseed meal feed (M1) significantly reduced the relative abundance of Fusobacteria and significantly increased the relative abundance of Firmicutes. In conclusion, when cottonseed meal in the feed was replaced by less than 51.7% soybean meal, it did not affect the growth performance of channel catfish, but it affected the skin color and flesh color of the fish.