Exposure to perfluorooctane sulfonate (PFOS) has been associated with lower bone density and the occurrence of osteoporosis in human studies, but the effects and mechanisms of PFOS induces bone loss is not well understood. Our research is aimed at examining the effects of PFOS on osteoblastic activity and investigating the toxicological mechanisms of PFOS-induced bone loss. Cell proliferation, ALP activity, bone nodule formation, ROS levels, and cell apoptosis were assessed after treating osteoblasts with different concentrations of PFOS. Through transcriptome analysis, the differentially expressed genes (DEGs) were screened and the biofunctions were elucidated by Kyoto Encyclopedia of Genes and Genomes (KEGG) and The Gene Set Enrichment Analysis (GSEA). Vation of important genes and protein expression was accomplished using RT-PCR and Western blot methods, respectively. The results show that PFOS can reduce bone formation markers and improve oxidative stress and cell apoptosis. The DEGs in PFOS-treated groups were involved in multiple pathways, including FoxO, HIF-1, Rap1, Hippo, and sphingolipid signaling. FoxO1 was validated as the key gene which regulates osteogenic differentiation and redox status. Our findings suggest that PFOS reduces bone formation through FoxO1-mediated oxidative stress and apoptosis, as well as inhibition of the OPG/RANKL and FoxO1/β-catenin pathways. It will be beneficial for early intervention or treatment of PFOS-induced bone loss, highlighting the importance of regulatory measures to limit human exposure to PFOS.