ABSTRACT The objective of this study was to investigate the effect of cottonseed peptides from in vitro degradation with protease, Enterococcus faecalis ( E. faecalis ), or both on the growth performance, nutrient digestibility, and health status of pigs. We used four diets containing non-degraded, protease-degraded, E. faecalis -degraded, or both-degraded cottonseed meal at 20% for growing phase (6 wk) and 16% for finishing phase (7 wk), corresponding to barrows, with six replicates of six pigs each. Results showed that the in vitro degradation of cottonseed meal increased proportions of peptides and organic acids and decreased nitrogen-free extract and phytate phosphorus. The degraded cottonseed meal increased feed intake, body weight gain, feed efficiency, apparent ileal digestibility of dry matter, energy, crude protein, Ile, Leu, Lys, Met, Cys, Ser, and Gly, and blood profiles of superoxide dismutase, glutathione peroxidase, catalase, IgA, and IgG. The diet containing cottonseed meal by dual degradation showed more pronounced effects on nutritional compositions, growth performance, nutrient digestibility, and serum parameters than the other diets. The data suggest that cottonseed meal dually degraded in vitro with enzymes and probiotics can improve its nutritional properties and application in animal feed. protease E , ) performance nondegraded, nondegraded non degraded, non-degraded proteasedegraded, proteasedegraded protease-degraded -degraded bothdegraded 20 6 wk 16 7 wk, barrows each nitrogenfree nitrogen free phosphorus intake gain efficiency matter energy protein Ile Leu Lys Met Cys Ser Gly dismutase peroxidase catalase IgA IgG compositions 2 1