Although chemotherapy-induced bone loss is well-recognized during breast cancer treatment, the underlying mechanism remains to be further elucidated, especially in patients with obesity. In this study, the objective was to investigate the impact of genomic silencing and pharmacological inhibition of S1P synthesis on bone loss in doxorubicin-induced obese breast cancer mice. In vitro study, upon the treatment of doxorubicin combined with palmitic acid, the S1P generated by 4T1 cells was significantly increased, resulting in an increase in osteoclastogenesis by activating the S1PR1/p-STAT3/NFATc-1 pathway in bone marrow-derived macrophages. In vivo study, pharmacological intervention with Sphingosine kinases (SPHK) antagonist SKI II or biological inhibition with SPHK1 and SPHK2 short hairpin RNA significantly reduced S1P production and rescued the obese breast cancer-bearing mice from doxorubicin-induced bone loss, manifested by the decreased osteoclastogenesis and recovered bone microarchitecture. Similarly, the administration of the S1PR1 antagonist FTY720 also alleviated bone loss in the breast cancer-bearing mice fed a high-fat diet. These studies indicate that genetic silencing and pharmacological inhibition can suppress S1P-dependent bone loss in doxorubicin-induced obese breast cancer mice. S1P shows promise as a potential drug target for preventing chemotherapy-induced bone loss in patients.