Abstract: Objective To investigate the role of Twist in regulating the proliferation, migration, and invasion of osteosarcoma cells with different levels of malignancy. Methods The baseline expressions of Twist in 3 different osteosarcoma cell lines (143B, MG63 and TE85) were detected using real-time PCR and Western blotting. The cells were infected with the recombinant adenoviruses Ad-Twist or Ad-siTwist for Twist overexpression or knockdown, respectively, and the cell growth curves were drawn to assess the cell proliferation. The migration abilities and invasiveness of the cells were evaluated using wound healing assay and Transwell assay. Luc-labeled 143B cells infected with Ad-Twist or Ad-siTwist were intrathecally injected to establish nude mouse models bearing osteosarcoma xenografts, in which the tumor formation was monitored using living body imaging technique. Results The baseline expressions of Twist in the 3 osteosarcoma cells were significantly higher than that in C3H10 cells (P<0.05). Twist expression was the highest in 143B cells followed by MG63 cells, and was the lowest in TE85 cells, indicating its positive correlation with the level of malignancy of the osteosarcoma cells. Ad- Twist or Ad- siTwist infection efficiently enhanced or lowered Twist expressions at both mRNA and protein levels in osteosarcoma cells (P<0.05). Twist overexpression resulted in enhanced proliferation, migration and invasion abilities of osteosarcoma cells, and Twist knockdown obviously inhibited the cell proliferation, migration and invasion. In nude mice, 143B cells with Twist overexpression showed accelerated tumor formation compared with the control cells, while Twist knockdown significantly inhibited the tumor formation ability of the cells. Conclusion Twist overexpression can promote the proliferation, migration, invasion and tumorigenicity of osteosarcoma cells.