MTMR14 is a phosphoinositide phosphatase, which has been reported to regulate the maintenance of normal muscle performance and aging in mice. However, the function of MTMR14 in mouse embryonic fibroblasts (MEFs) remains largely unknown. In this study, we established MTMR14 WT and KO MEFs and showed that MTMR14 is localized in whole MEFs, with higher level in nucleus and lower in cytoplasm, partially overlapping with mitochondrial. Compared with the WT control, MTMR14 KO MEFs exhibit a higher proliferation rate and more obvious autophagy. Furthermore, we demonstrate that KO of MTMR14 significantly decreased the mRNA levels of p21 and p27, while increased those of cyclinD and cyclinE. Upon (insulin-like growth factor) IGF stimulation, we also found KO of MTMR14 enhanced the phosphorylation levels of AKT and ERK in MEFs. Based on these findings, we propose that defect of MTMR14 promotes autophagy and cell proliferation in MEFs.