Growth Arrest Specific 5 (GAS5), a long noncoding RNA (lncRNA), functions as a tumor suppressor in multiple cancers. However, its function, downstream targets and upstream regulatory mechanism are still obscure in osteosarcoma cells. Here, we discovered that GAS5 was downregulated in cancerous osteosarcoma tissues and cells. Using a microarray analysis, we identified that GAS5 can regulate the expression of TPS3, Bax, Bim, DDB2, TGFB and ROS1 in osteosarcoma cells. Specifically, GAS5 overexpression in the U2OS osteosarcoma cell line induced TPS3, Bax and Bim levels but inhibited DDB2, TGFB and ROS1 expression, resulting in the inhibition of cell proliferation, invasion, colony formation and in vivo tumor formation. By analyzing the GAS5 promoter region (-2000), we identified several potential transcription factor-binding sites including NF-kappa B, IK-1, AP-1, SP1 and IRF1. By individually knocking down these transcription factors, we found that only knockdown of IRF1 affected GAS5 expression. Using immunoprecipitation (IP), mass spectrometry assays, and co-IP assays, we identified that IRF1 formed a transcriptional complex with Histone Deacetylase 1 and 2 (HDAC1/2) and C-terminal binding protein 1 (CtBP1). Functional analyses indicated that the CtBP1-HDAC1/2-IRF1 complex specifically bound to the GAS5 promoter and regulated its expression and downstream events. Knockdown of CtBP1 or overexpression of IRF1 in osteosarcoma cells can significantly reverse their oncogenic phenotypes. Altogether, our results indicated that the CtBP1-HDAC1/2-IRF1 transcriptional complex inhibited GAS5-mediated signaling in osteosarcoma cells, and it might be a potential therapeutic target for osteosarcoma treatment.