Despite recent advances in diagnosis and treatment, osteosarcoma remains as the most common bone cancer in children and is associated with poor prognosis. Growing evidence has supported dysregulation of threonine and tyrosine protein kinase (TTK) expression as a hallmark of multiple cancers, however, its function in osteosarcoma remains to be elucidated. In the present study, we found that TTK was frequently overexpressed in osteosarcoma and associated with increased tumour growth and progression. Moreover, using both in vitro and in vivo assays, we provided evidence that TTK level was regulated by a molecular chaperone, heat shock protein 90 (Hsp90). Hsp90 directly interacted with TTK and prevents proteasome-dependent TTK degradation, leading to the accumulation of TTK in osteosarcoma cells. Elevated TTK promoted cancer cell proliferation and survival by activating cell-cycle progression and inhibiting apoptosis. Consistently, depletion of TTK by Hsp90 inhibition induced cell-cycle arrest, generated aneuploidy and eventually resulted in apoptotic cancer cell death. Together, our study revealed an important Hsp90-TTK regulatory axis in osteosarcoma cells to promote cancer cell growth and survival. These findings expand our knowledge on osteosarcoma pathogenesis and offer novel therapeutic options for clinical practice.