Myofibroblasts combine features of fibroblasts and smooth muscle cells, and they are reactive cells present under injury conditions. This study was performed to explore the mechanism that methylenetetrahydrofolate dehydrogenase/cyclohydrolase 2 (MTHFD2) mediated m6A modification in sepsis-induced AKI (SAKI) through regulating the collagen accumulation in myofibroblasts. Gene expression microarrays related to SAKI were obtained from the GEO database, and the hub protein involved was screened using PPI. The SAKI mice were induced by cecal ligation and puncture (CLP). MTHFD2 expression was significantly elevated in the kidneys of CLP-induced mice, and SAKI was ameliorated by knocking down MTHFD2 in kidney tissues. MTHFD2 promoted N-methyladenosine (m6A) modification in kidney tissues of CLP-induced mice by increasing the content of methylated donor s-adenosylmethionine (SAM). MTHFD2 enhanced LOX mRNA stability in an m6A modification-dependent manner, thereby promoting its expression. Knockdown of MTHFD2 inhibited collagen accumulation in myofibroblasts, whereas overexpression of LOX accelerated fibrosis and SAKI in mice in the presence of sh-MTHFD2. In conclusion, our results show that MTHFD2 promotes LOX expression in an m6A-dependent manner, thereby mediating SAKI progression.