MicroRNAs (miRNAs) and DNA mismatch repair (MMR) have been linked to human cancer progression. Human mutL homolog 1 (hMLH1), one of the core MMR genes, defects in lung cancer development. However, the interaction between miRNAs and MMR genes and their regulatory effect on cell cycle remain poorly understood. In this study, we investigated the role of miR-31-5p in hMLH1 gene expression and the effect of miR-31-5p on cell cycle in non-small cell lung cancer (NSCLC). We found that miR-31-5p was inversely correlated with hMLH1 expression in NSCLC cell lines and hMLH1 was a direct target of miR-31-5p. Knockdown of miR-31-5p induced a cell cycle arrest at G2/M phase and increased hMLH1 protein expression in NSCLC cells. Conversely, overexpression of miR-31-5p significantly induced cell cycle arrest at S phase and decreased hMLH1 protein expression. Furthermore, knockdown of hMLH1 upregulated miR-31-5p expression and caused cell cycle arrest at S phase. Data from this study revealed that miR-31-5p modulates cell cycle by targeting hMLH1 protein at the posttranscriptional level in NSCLC, which may represent a novel therapy strategy for lung cancer by targeting miR-31-5p.