The aim of this study was to investigate the effects of bufalin on the mammalian target of rapamycin (mTOR/p70S6 kinase (p70S6K) signaling pathway and cell apoptosis in orthotopically transplanted tumors in nude mice. The mice were inoculated with human esophageal squamous cell carcinoma (ESCC) ECA109 cells in order to establish a model of orthotopicall transplanted ESCC tumors. The mice are administered low, medium and high doses of bufalin (0.5, 1.0 and 1.5 mg/kg) or rapamycin, or a combination of both. After the tumors were removed, the mRNA expression levels of mTOR, p70S6K, eukaryotic translation initiation factor 4E-binding protein 1 (4EBP1), cellular inhibitor of apoptosis protein 1 (cIAP1) and caspase-3 were detected by RT-PCR. In addition, we performed western blot analysis and immunohistochemical analysis to determine the protein expression of mTOR, p70S6K, 4EBP1, cIAP1, active caspase-3, Bcl-2 and Bad in the tumor tissue. The results revealed that bufalin exerted a significant anti-tumor effect in the nude mice with ESCC orthotopically transplanted tumors. This was shown by the decrease in the expression of mTOR, p70S6K and 4EBP1, which suggested that bufalin may possibly be used to inhibit tumor growth via the inhibition of the activation of p70S6K and 4EBP1. We also found that bufalin decreased the expression of cIAP1 and Bcl-2, and increased that of active caspase-3 and Bad, thus indicating that bufalin promoted apoptosis. Thus, our findings suggest that bufalin promotes tumor cell apoptosis, and this may be one of the important anti-tumor mechanisms of action of bufalin.