Abstract: Objective To investigate the effect of miR-101 expression on invasion and autophagy of osteosarcoma cell line MG-63. Methods qRT-PCR was applied to detect miR-101 expression in human osteosarcoma tissues, MG-63 cells and osteoblasts. Western blot was used to measure the protein expression of Beclin1 and LC3B. Moreover, MG-63 cells were randomly divided into three groups by means of lipofectamine transfection: miR-101 mimics group, negative control group and blank control group. After transfection, miR-101 expression was detected by qRT-PCR, protein levels were measured by Western blot, and Transwell migration assay was used to examine the invasive capacity of MG-63 cells. Results miR-101 expression levels in osteosarcoma tissues and MG-63 cells were significantly decreased, compared with those in normal bone tissues, adjacent bone tissues and osteoblast (P<0.01). Compared with blank control group, miR-101 expression in miR-101 mimics group was up-regulated significantly by 255%, but the proteins expression of Beclin1 and LC3B were obviously decreased (P<0.01). Meanwhile, Transwell migration assay showed that the numbers of migrated cells in miR-101 mimics group were decreased by 60% and 67.67% compared with those in negative control group and blank control group, respectively (P<0.01). Conclusion miR-101 expression was down-regulated significantly in osteosarcoma tissues and MG-63 cells, which may be involved in the occurrence and development of osteosarcoma. miR-101 could dramatically inhibit Beclin1 and LC3B protein expression and retard the invasion of osteosarcoma MG-63 cells, and the mechanism may be achieved by inhibiting autophagy.