Osteosarcoma (OS) is a primary malignant bone neoplasm with high frequencies of tumor metastasis and recurrence. Although the Akt/PKB signaling pathway is known to play key roles in tumorigenesis, the roles of cyclin-dependent kinase-like 3 (CDKL3) in OS progression remain largely elusive. We have demonstrated the high expression levels of CDKL3 in OS human specimens and comprehensively investigated the role of CDKL3 in promoting OS progression both in vitro and in vivo. We found that CDKL3 regulates Akt activation and its downstream effects, including cell growth and autophagy. The up-regulation of CDKL3 in OS specimens appeared to be associated with Akt activation and shorter overall patient survival (P = 0.003). Our findings identify CDKL3 as a critical regulator that stimulates OS progression by enhancing Akt activation. CDKL3 represents both a biomarker for OS prognosis, and a potential therapeutic target in precision medicine by targeting CDKL3 to treat Akt hyper-activated OS.