Osteosarcoma is a common bone tumor which is affected by E2, the most representative estrogen. Gene regulation function of E2 is highly dependent on estrogen receptor. The purpose of this study was to explore the gene regulation patterns of E2 through estrogen receptor α (ESR1) in osteosarcoma based on the combined analysis of ChIP-seq and gene microarray. All of the datasets were downloaded from the Gene Expression Omnibus (GEO). Differential expression genes (DEGs) in E2 treated U2OS cells expressing ESR1 (U2OS-ERα) compared with those treated with vehicle were obtained based on R programming software. ESR1-specific binding sites (peaks) in E2 treated U2OS cells were identified through MACS. Overlaps between DEGs and ESR1 target genes which contained peaks in promoters were considered as reliable E2-mediated genes through ESR1 in osteosarcoma. Moreover, we conducted miRNA-Gene regulation analysis for those genes through miRWalk database to identify potential therapeutic targets for the genes. Functional enrichment analysis of DEGs indicated their potential involvement in cancer, and cell activity-related processes. Fifteen overlaps were identified between DEGs and target genes of ESR1, of which 12 were found to be regulated by miRNA. Several known estrogen response genes and novel genes were obtained in this study and they might provide potential therapeutic targets for osteosarcoma.