Simple SummaryOsteosarcoma is the predominant form of primary bone cancer and outcomes for patients with metastatic disease have not improved for several decades. Recent genomic and pharmacological studies have implicated dysregulated histone deacetylases as druggable targets to treat several types of cancers, including osteosarcoma. This study aimed to assess the inhibitory effects of 4SC-202, a next-generation inhibitor for class I histone deacetylases, on human osteosarcoma cell growth in vitro and in vivo. We demonstrated that the anti-tumor effect of 4SC-202 involves combined cell-cycle arrest and apoptosis, as well as a reduction in cell invasion and migration. Moreover, 4SC-202 revised the global transcriptome and induced distinct signatures of gene expression in vitro. Additionally, 4SC-202 decreased tumor growth of established human tumor xenografts in immunodeficient mice in vivo. Our study suggests that 4SC-202 may be exploited as a valuable drug to promote more effective treatment of patients.Dysregulation of histone deacetylases (HDACs) is associated with the pathogenesis of human osteosarcoma, which may present an epigenetic vulnerability as well as a therapeutic target. Domatinostat (4SC-202) is a next-generation class I HDAC inhibitor that is currently being used in clinical research for certain cancers, but its impact on human osteosarcoma has yet to be explored. In this study, we report that 4SC-202 inhibits osteosarcoma cell growth in vitro and in vivo. By analyzing cell function in vitro, we show that the anti-tumor effect of 4SC-202 involves the combined induction of cell-cycle arrest at the G2/M phase and apoptotic program, as well as a reduction in cell invasion and migration capabilities. We also found that 4SC-202 has little capacity to promote osteogenic differentiation. Remarkably, 4SC-202 revised the global transcriptome and induced distinct signatures of gene expression in vitro. Moreover, 4SC-202 decreased tumor growth of established human tumor xenografts in immunodeficient mice in vivo. We further reveal key targets regulated by 4SC-202 that contribute to tumor cell growth and survival, and canonical signaling pathways associated with progression and metastasis of osteosarcoma. Our study suggests that 4SC-202 may be exploited as a valuable drug to promote more effective treatment of patients with osteosarcoma and provide molecular insights into the mechanism of action of class I HDAC inhibitors.