A 12‐week feeding trial was conducted to investigate the protective effects of arginine on the intestinal health of juvenile turbot (Scophthalmus maximus L.) fed high doses of dietary soybean meal. Sextuple replicates of 30 fish were fed each of four isonitrogenous and isolipidic experimental diets: fish‐meal‐based diet (FM); FM with 40% fish meal protein replaced by soybean meal protein (SBM); SBM+1% arginine (ARG1) and SBM+2% arginine (ARG2). Turbot‐fed SBM showed typical soybean meal‐induced enteropathy, characterized by an increase in the thickness of lamina propria, as well as significant decreases in the absorptive surface and activities of intestinal brush border membrane enzymes and Na+, K+‐ATPase. On the contrary, fish‐fed ARG1 showed enhanced intestinal mucosal barrier function in terms of the enhanced gene expression of anti‐inflammatory cytokine and barrier‐forming tight junction proteins, as well as depressed gene expression of pro‐inflammatory cytokines and pore‐forming tight junction proteins. Fish‐fed AGR2 showed intermediate intestinal performances between SBM and AGR1. Dietary arginine (1%) also significantly regulated the expression of AMP‐activated protein kinase α1 (AMPKα1), myosin light chain kinase (MLCK) and nuclear transcription factor‐κB p65 (NF‐κB p65), and these regulations correlated well with its regulations on intestinal mucosal barrier related genes at all sampling time‐points. In conclusion, arginine supplementation (1%) in diet for turbot mitigated the soybean meal‐induced enteropathy by enhancing the intestinal mucosal barrier function. The activation of AMPKα1 signalling molecule as well as the suppression of NF‐κB p65 and MLCK signalling molecules may mediate the beneficial effects of arginine.