Our previous study has demonstrated that supplementation of yeast β-glucan improves intestinal health in pearl gentian grouper (Epinephelus lanceolatus♂ × Epinephelus fuscoguttatus♀), accompanied by the activation of the mitogen-activated protein kinase (MAPK) signaling pathway. In this study, we investigated the effects of perturbing p38 MAPK activity using an inhibitor on the intestinal health of β-glucan-injected pearl gentian grouper to elucidate the potential molecular mechanism underlying the protective effects of β-glucan on the fish gut. The pearl gentian grouper was categorized into four groups: PBS injected (CD group), β-glucan injected at a dose of 80 mg/kg (βG group), p38 MAPK inhibitor SB203580 injected at a dose of 1 mg/kg (SB203580 group), and a combination of β-glucan (80 mg/kg) and SB203580 (1 mg/kg) injected together (βG + SB203580 group). The results revealed that the introduction of SB203580 significantly suppressed the β-glucan-induced increase in p38α and p38β mRNA expression, as well as the phosphorylation of p38 MAPK. Both the βG group and SB203580 group exhibited reduced plica height and muscularis thickness. The βG + SB203580 group displayed a significant reduction in mucin cell level; interleukin 1β (il1β) mRNA expression; induced nitric oxide synthase, tumor necrosis factor α, and IL1β concentration; catalase and total antioxidant capacity activities. Additionally, there was a significant increase in the levels of intestinal malondialdehyde in the βG + SB203580 group compared to the βG group. The inhibition of the p38 MAPK signaling halted the trend of apoptosis-related caspase molecular expression induced by β-glucan. In conclusion, β-glucan injection resulted in elevated levels of mucous cells, nonspecific immunity, antioxidant capacity, and anti-apoptosis in grouper by modulating the p38 MAPK pathway. This study offers insights into the potential molecular mechanism underlying the protective effects of β-glucan on intestinal health in pearl gentian grouper.