Rapamycin may serve as a new anti-osteosarcoma (OSA) agent due to its ability to inhibit the metastatic behavior of OSA. However, only limited benefit is observed in rodent studies and clinical trials using rapamycin as a single agent in the treatment of OSA. The target of rapamycin, mammalian target of rapamycin has multiple biological functions and may be linked with the kinases that mediate the phosphorylation of cyclic AMP-responsive element-binding (CREB) protein, an import factor in tumor progression. By employing an OSA cell line MG-63, we investigated how rapamycin regulates the phosphorylation of CREB (pCREB) at Ser133 and the expressions of two putative CREB targets, B-cell lymphoma 2 (Bcl-2) and vascular endothelial growth factor-A (VEGF-A). Under normoxia, we found that rapamycin (100 nM) induced an increase of pCREB that was prevented by mitogen-activated protein kinase (MEK)/extracellular signal-regulated kinase (ERK) inhibitor U0126 or cAMP-dependent protein kinase (PKA) inhibitor H89. However, H89 enhanced Akt phosphorylation and did not decrease the cell viability upon rapamycin treatment. In contrast, U0126 did not enhance Akt phosphorylation and decreased the cell viability upon rapamycin treatment. Moreover, U0126 prevented the rapamycin-induced increase of Bcl-2 and VEGF-A levels. Under hypoxia, rapamycin effectively prevented the hypoxia-induced increase of pCREB, Bcl-2, and VEGF-A. Our study demonstrated that rapamycin might be less effective in treating OSA cells under normoxia and provided the rationale for a combination of rapamycin and MEK/ERK inhibitor in the treatment of OSA.