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Objective: To investigate the effect of Aurora kinase B (AURKB) silencing-induced autophagy on apoptosis of osteosarcoma 143B cells and the underlying molecular mechanisms., Methods: Human osteosarcoma 143B cells were transfected with Lv/shAURKB or the negative control vector Lv/shScrambled followed by treatment with chloroquine (CQ) for 24 h. Western blotting was performed to detect the protein expression levels of AURKB, P62, LC3, cleaved caspase-3, Bcl-2, and P-ULK1 Ser555 . Transmission electron microscopy and LC3 dual-label fluorescence method were used to trace the autophagosomes in 143B cells to assess cell autophagy, and the cell apoptosis was detected using flow cytometry and TUNEL assay. Co-immunoprecipitation assay was used to detect the interaction between AURKB and ULK1., Results: The ratio of autophagy-related proteins LC3 II/I and the number of autophagosomes were significantly increased in 143B cells after transfection with Lv/shAURKB ( P < 0.05), which significantly increased the expression of cleaved caspase-3 and reduced the expression of Bcl-2 ( P < 0.05). Combined treatment of the cells with Lv/shAURKB and the autophagy inhibitor chloroquine obviously restored the expressions of caspase-3 and Bcl-2 ( P < 0.05). Transfection with Lv/shAURKB significantly increased the apoptosis rate of 143B cells ( P < 0.05), and this effect was significantly antagonized by combined treatment with chloroquine ( P < 0.05). AURKB silencing strongly activated the phosphorylation of the autophagy-initiating protein ULK1 Ser555 in 143B cells ( P < 0.05). The results of co-immunoprecipitation assay confirmed when AURKB was immunoprecipitated, ULK1 also precipitated., Conclusions: Silencing AURKB can induce autophagy by activating ULK1 Ser555 phosphorylation to promote apoptosis in 143B cells.