Objective: CLEC-2 signaling pathway is involved in the regulation of autophagy in megakaryocytes. However, the mechanism has not been fully elucidated. In this study, CLEC-2 deficient mice were used to detect whether CLEC-2 plays an important role in autophagy. Methods: Megakaryocytes were isolated and cultured from 14.5 day pregnant mouse embryos. The expression of CLEC-2 and CD41 in megakaryocytes were detected. After rapamycin treatment, the expression of autophagy protein was detected. Results: We found that autophagy gene was highly expressed in megakaryocytes derived from fetal liver. After rapamycin treatment, the expression of CLEC-2 was decreased, indicating that CLEC-2 was involved in the regulation of autophagy. Compared with wild-type mice, the expression of CD41 and CD61 in megakaryocytes of rapamycin knockout mice was significantly decreased. In vivo, rapamycin can significantly enhance the expression of LC3 protein in knockout mice. The defect in CLEC-2 can significantly reduce the number of polyploid megakaryocytes caused by autophagy disorder. And it also reduced the expression of cyclin D1, cyclind2 and p21 in megakaryocyte derived from fetal liver induced by rapamycin. Conclusion: CLEC-2 plays an important role in the regulation of autophagy of megakaryocytes.