Emerging evidence has suggested that glycolysis is enhanced in cancer-associated fibroblasts (CAF), andmiR-186 is downregulated during the CAF formation. However, it is not clear whether miR-186 is involved in theregulation of glycolysis and what the role of miR-186 plays during the CAF formation. In this study, quantitativePCR analysises show miR-186 is downregulated during the CAF formation. Moreover, miR-186 targets the 3’UTR of Glut1, and its overexpression results in the degradation of Glut1 mRNA, which eventually reduces thelevel of Glut1 protein. On the other hand, knockdown of miR-186 increased the expression of Glut1. Both timecourse and dose response experiments also demonstrated that the protein and mRNA levels of Glut1 increaseduring CAF formation, according to Western blot and quantitative PCR analyses, respectively. Most importantly,besides the regulation on cell cycle progression, miR-186 regulates glucose uptake and lactate production whichis mediated by Glut1. These observations suggest that miR-186 plays important roles in glycolysis regulation aswell as cell cycle checkpoint activation.