The present study was conducted to investigate the effects of dietary fermented cottonseed meal (FCSM) substitution for fish meal on intestinal enzymatic activity, inflammatory and physical-barrier-related gene expression, and intestinal microflora of juvenile golden pompano. The 375 golden pompanos were divided into 15 groups of 25 fish each, with three replicates for each experimental group. The fish were fed five experimental diets (0 (FM), 12.5% (CSM12.5), 25% (CSM25), 50% (CSM50), and 100% (CSM100) substitution levels) for 8 weeks. The fish were reared and fed the experimental diets under a natural-day light cycle. Compared with the control group, the activities of AMY (amylase) enzymes in the CSM12.5 group and all other groups were elevated (p < 0.05). The CSM25 group exhibited a considerable up-regulation of IL-10 (Interleukin-10) expression relative to the FM group (p < 0.05). With an increase in dietary FM substitution with FCSM from 0 to 25%, the relative expressions of NF-κB (Nuclear factor kappa-B), IL-1β (Interleukin-1 beta), and IL-8 (Interleukin-8) were down-regulated. In this study, the relative expressions of ZO-1 (zonula occluden-1) and Occludin were up-regulated, and those of Claudin-3 and Claudin-15 significantly up-regulated, when the FCSM substitution ratio was 25%. The results of high-throughput sequencing of the intestinal microflora showed that ACE indices the lowest in the CSM25 group, which was significantly different from those in the CSM100 group (p < 0.05). The CSM50 group had the highest Shannon and Simpson indices and the highest community diversity. In addition, replacing a high percentage of fish meal with FCSM can negatively affect the intestinal flora of fish. In this study, the 25% substitution ratio improved nutrient absorption, reduced intestinal inflammation, improved intestinal physical barrier damage, did not affect intestinal microecology, and had no adverse effects on fish. However, substitution of a high proportion of FM with FCSM negatively affects the intestinal microflora and nutrient absorption capacity of fish.