Background: Human U2OS osteosarcoma cells were first derived from a tibial osteosarcoma of a teenage girl. They are a good cell model for osteosarcoma research in vitro. We compared the expression profiles of osteosarcoma cells after they were treated with estradiol and glucocorticoid, respectively. Materials and Methods: We used published microarray data to compare the expression profiles of human osteosarcoma cells treated with estradiol and glucocorticoid. We investigated their differences and similarities with various bioinformatics tools. Oncogenes and tumor-suppressor genes differentially expressed after the hormone treatments were identified using online databases. The expression levels of cellular markers for proliferation and apoptosis were also compared before and after treatment with estradiol or glucocorticoid. Results: Genes in human osteosarcoma cells differentially expressed after treatment with estradiol or glucocorticoid were detected. Our analysis showed that their similarity is more functionally prominent than their difference. Both estradiol and glucocorticoid can inhibit purine metabolic and biosynthetic pathway in human osteosarcoma cells. We also identified oncogenes and tumor-suppressor genes among the differentially expressed genes. The functional enrichment analyses of the identified cancer-associated genes suggests that estradiol has antagonistic effects on regulation of cell proliferation, while glucocorticoid can both arrest the cell cycle and prompt apoptosis. The effect of estradiol or glucocorticoid treatment on expression levels of cellular markers for proliferation and apoptosis support this argument. Conclusion: Our study suggests that glucocorticoid is more efficient in controlling osteosarcoma cell proliferation and apoptosis than estradiol.