Objective: To investigate the effect of miR-26 targeting methylenetetrahydrofolate reductase (MTHFR) by JAK/STAT signaling pathway on invasion and proliferation of lung cancer. Methods: The expression of MTHFR in lung cancer tissues was detected by immunohistochemistry. The expression of miR-26 in lung cancer cell line was detected by PCR. The effect of miR-26 on the expression of MTHFR was examined by the dual luciferase gene system. Effect of miR-26 on the proliferation ability of H1650 were detected by tumor in ball assays. Transwell invasion assay was used to detect the ability of invasion in the lung cancer cell line H1650 after overexpression of miR-26. Detection of reactive oxygen species in lung cancer cells by CM-H2DCFDA fluorescent staining cells. Western blot was used to detect the protein expression of JAK/STAT signal pathway after overexpression of miR-26. Results: The expression of miR-26 in lung cancer tissues was significantly lower than that in normal lung tissues. The dual luciferase reporter gene system showed that miR-26 could directly regulate the transcriptional activity of MTHFR after expression of miR-26, the proliferation and invasion ability of lung cancer cell H1650 were significantly decreased, and the reactive oxygen level in lung cancer cells decreased. After expression of miR-26, the expression level of MTHFR was down-regulated, and p-JAK and p-STAT protein were down-regulated. Conclusion: miR-26 targeted MTHFR can regulate the proliferation and invasion of lung cancer cells by the JAK/STAT pathway.