Ulcerative colitis (UC) represents a significant challenge to global health, underscoring the importance of developing novel alternative anti-colitis agents. Inhibition of the NLRP3 inflammasome in macrophages has emerged as a potential therapeutic strategy for UC. Pulchinenoside B4 (PB4) is a major component of traditional medicinal plants that demonstrated to possess promising anti-inflammatory properties. The aim of the present study was to assess whether PB4 alleviates dextran sodium sulfate (DSS)-induced colitis by inhibiting the NLRP3 inflammasome in macrophages and its potential molecular mechanism. We constructed DSS-induced colitis in C57BL/6 mice, and isolated mouse intestinal macrophages and epithelial cells to investigate the effect of PB4 on NLRP3 inflammasome, and confirmed our findings in DSS-induced NLRP3-/- mice. In addition, we constructed lipopolysaccharides (LPS)-induced macrophages in vitro and identified the target and molecular mechanism of PB4 through biolayer interference (BLI) and cell thermal migration (CETSA) in conjunction with dss induced macrophage-specific CD1d depletion (CD1d-/-) colitis. This study showed that PB4 had a strong anti-inflammatory effect on WT mice induced by DSS, but the protective effect on NLRP3-/- mice was no longer enhanced. Interestingly, PB4 inhibited the activation of NLRP3 inflammasome in colon macrophages without affecting intestinal epithelial cells. Mechanistically, PB4 may target CD1d, thereby reducing the AKT-STAT1-PRDX1-NF-κB signaling pathway and ultimately inhibiting the activation of the NLRP3 inflammasome. Macrophage-specific CD1d loss has been shown to reverse the protective effects of PB4. These findings have paved the way for the development of CD1d/NLRP3-based novel anti-colitis agents and will facilitate the future clinical translation of the plant-derived drug PB4.