ObjectiveTo investigate the regulatory effect of Mankuining Formula （MKNF） on the gut microbiota and the NOD-like receptor （NLR）P3/Caspase-1/gasdermin D （GSDMD） pyroptosis pathway-mediated inflammation in dextran sulfate sodium （DSS）-induced ulcerative colitis （UC） mice. MethodSixty SPF C57BL/6 mice were randomly divided into a blank group， a model group， a MKNF group （20 g·kg^-1）， and a mesalazine group （0.266 g·kg^-1）， with 15 mice in each group. The UC model was induced in mice by freely drinking a 3% DSS solution for 7 days. After 12 hours of modeling， the treatment groups received daily oral administration， while the other groups received an equal volume of normal saline by gavage. Daily body weight and disease activity index （DAI） were recorded. On the 8th day， mice were euthanized after anesthesia， and the colon and feces were collected. The colon length was measured， and histopathological changes were observed after hematoxylin-eosin （HE） staining. Tumor necrosis factor-α （TNF-α）， interleukin-1β （IL-1β）， and interleukin-18 （IL-18） levels in the colon were detected by enzyme-linked immunosorbent assay （ELISA）. The differences in gut microbiota among the groups were analyzed using 16S rRNA sequencing technology. The protein content of NLRP3/Caspase-1/GSDMD in colon tissues was detected by Western blot. ResultCompared with the blank group， mice in the model group showed increased DAI （P<0.01）， shortened colon length （P<0.01）， severe colon mucosal damage， elevated levels of TNF-α， IL-1β， and IL-18 （P<0.01）， increased protein content of NLRP3/Caspase-1/GSDMD in colon tissues （P<0.01）， altered gut microbiota structure with decreased abundance of Actinobacteria， Bacteroidetes， and Proteobacteria， and increased abundance of Firmicutes at the phylum level. At the genus level， there was a decrease in Lactobacillus， Alloprevotella， and Yersinia， and an increase in Bacteroides， Bacillus， and Lachnospiraceae_NK4A136. Compared with the model group， the MKNF group and the mesalazine group showed a significant reduction in DAI after the 3^rd day （P<0.01）， a significant increase in colon length （P<0.01）， alleviated colon inflammation and mucosal structural damage， and decreased TNF-α， IL-1β， and IL-18 levels in the colon （P<0.01）， reduced protein content of NLRP3/caspase-1/GSDMD in colon tissue （P<0.05， P<0.01），an increase in the abundance of Proteobacteria and Bacteroidetes， and a decrease in Firmicutes at the phylum level. ConclusionMKNF can alleviate UC-induced colonic inflammation， reduce colon damage， and improve dysbiosis of the gut microbiota by inhibiting the classical pyroptosis pathway.