Accumulating evidence suggests that neuropathic pain (NP) is closely connected to the metabolic disorder of gut microbiota, and natural products could relieve NP by regulating gut microbiota. The purpose of this study is to investigate the important regulatory effects of osthole on gut microbiota and serum metabolites in mice with chronic constriction injury (CCI). Mice's intestinal contents and serum metabolites were collected from the sham group, CCI group, and osthole treatment CCI group. The 16S rRNA gene sequencing was analyzed, based on Illumina NovaSeq platform, and ANOVA analysis were used to analyze the composition variety and screen differential expression of intestinal bacteria in the three groups. Ultra-high-performance liquid chromatography-quadrupole time of flight-tandem mass spectrometry (UHPLC-Q-TOF-MS) was used for analyzing the data obtained from serum specimens, and KEGG enrichment analysis was used to identify pathways of differential metabolites in the treatment of neuralgia mice. Furthermore, the Pearson method and Cytoscape soft were used to analyze the correlation network of differential metabolites, gut microbiota, and disease genes. The analysis results of 16S rRNA gene sequencing displayed that , , and were highly correlated with NP after osthole treatment at the phylum level. , , , , , and exhibited higher relative abundance and were considered important microbial members at genus level in neuralgia mice. Serum metabolomics results showed that 131 metabolites were considered to be significantly different in the CCI group compared to the sham group, and 44 metabolites were significantly expressed between the osthole treatment group and the CCI group. At the same time, we found that 29 differential metabolites in the two comparison groups were overlapping. Integrated analysis results showed that many intestinal microorganisms and metabolites have a strong positive correlation. The correlation network diagram displays that 10 genes were involved in the process of osthole alleviating NP through a metabolic pathway and gut microbiota, including IGF2, GDAP1, MYLK, IL18, CD55, MIR331, FHIT, F3, ERBB4, and ITGB3. Our findings have preliminarily confirmed that NP is closely related to metabolism and intestinal microbial imbalance, and osthole can improve the metabolic disorder of NP by acting on gut microbiota.