Background: The primary reason for the development of chemotherapeutic intestinal mucositis induced by 5-fluorouracil (5-FU) is the activation of macrophages in the mucosa. This study aimed to explore how berberine can alleviate inflammation in macrophages and the resulting intestinal mucosal inflammation in mice induced by 5-FU. Methods: Tohoku Hospital Pediatrics-1 (THP-1) cell inflammatory response and mouse intestinal mucositis were induced by 5-FU and treated with berberine. The levels of inflammation-related factors and autophagy related proteins in THP-1 cells were detected by reverse transcription quantitative polymerase chain reaction (RT-qPCR) and western blotting. The concentrations of double-stranded DNA (dsDNA) and interleukin-1β (IL-1β) in mice serum and the small intestine hematoxylin-eosin (HE) staining were used to evaluate intestinal mucosal damage. Immunoglobulin A (IgA), an indicator of mucosal immunity, was detected in mice serum by enzyme-linked immunosorbent assay (ELISA). We used quantitative polymerase chain reaction (qPCR) to detect the relative contents of four important strains (Bifidobacterium, Lactobacillus, Escherichia coli, and Enterococcus) in the colon contents of mice. Additionally, we employed liquid chromatograph-mass spectrometer/mass spectrometer (LC-MS/MS) technique to measure the concentrations of three main short-chain fatty acids (acetic acid, propionic acid, and butyric acid) in the mices plasma. Furthermore, we employed ultra-performance liquid chromatography-quadrupole-time of flight-mass spectrometer (UPLC-Q Tof-MS) technique to analyze the non-targeted metabolomics of mouse serum. Results: Berberine has been found to inhibit the expressions of NOD-Like Receptor Thermal Protein Domain Associated Protein 3 (NLRP3), Caspase-1, and IL-1β in THP-1 cells (p < 0.05). Additionally, it effectively suppresses the expression of autophagy related proteins LC3 and Beclin-1 in THP-1 cells (p < 0.05). Furthermore, in a mouse model study, berberine significantly enhances the levels of beneficial bacteria Bifidobacterium and Lactobacillus, as well as the concentrations of three main short-chain fatty acids (SCFAs) in the plasma of mice (p < 0.05). Moreover, it reduced the concentration of pro-inflammatory factor dsDNA and increased the mucosal immunity index IgA concentration in blood (p < 0.05). The untargeted metabolomics results demonstrated that berberine could regulate the inflammatory response of mice by impacting the metabolism of taurine, glycerol phospholipid, arachidonic acid, and primary bile acid biosynthesis. Conclusions: Berberine has demonstrated its ability to effectively suppress the inflammatory reaction of THP-1 cells induced by 5-FU. Furthermore, it also influences the “intestinal flora-metabolite-inflammation” pathway by regulating the composition of the intestinal flora, increasing the production of SCFAs, reducing the expression of inflammatory factors, and preserving the structural integrity of the intestinal mucosa.