Osteosarcoma (OS) is the most domain primary malignant bone tumor. Treatment resistances and metastases result in a decreasing 5-year overall survival rate of OS. However, Rho GTPase-activating protein 44 (ARHGAP44) has not been well studied in OS. The OS patient data were obtained from Therapeutically Applicable Research to Generate Effective Treatments and Gene Expression Omnibus databases. We utilized Survival and Survminer package for survival analysis based on Kaplan–Meier method. The association between ARHGAP44 expression with the prognosis of OS was determined by Wilcoxon rank-sum test and multivariate Cox regression analysis. The real-time polymerase chain reaction and western blotting were conducted to validate the results. Gene set enrichment analysis was done to find significant Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways. The OS sample ARHGAP44 expression level was significantly higher than that in normal samples, which was validated in cell lines. High ARHGAP44 expression was associated with metastasis of OS. The OS patients with high ARHGAP44 expression had worse prognosis compared with low ARHGAP44 expression OS patients. In total, 10 KEGG pathways significantly activated in high ARHGAP44 expression OS patients, such as Hedgehog signaling pathway, Steroid biosynthesis, and so on. In summary, high ARHGAP44 expression was closely correlated with the metastasis and poor prognosis of OS. ARHGAP44 was a potential prognostic biomarker for OS.