The purpose of this study was to develop an alternative submicron emulsion containing three bufadienolides for oral administration and evaluate its preclinical stability, efficacy, and toxicity. The bufadienolide-loaded oral submicron emulsion (BU-OE) was prepared by high-pressure homogenization. The storage stability, in vitro cytotoxicity, in vivo antitumor efficacy, acute toxicity, and long-term toxicity of BU-OE were investigated in detail to evaluate the formulation. The stability study suggested that BU-OE was stable at room temperature and could be stored for at least 18 months at 6±2 °C. The cytotoxicity test revealed that BU-OE had marked cytotoxic activities against cancer cells, but no evident inhibitory effects on normal cells. Likewise, BU-OE exhibited significant antitumor efficacy against Hep G2, HCT-8, and EC9706 cell lines and a slight inhibitory effect on BGC 803 cell line in nude mice, while comparable antitumor activity with fluorouracil injection. The LD50 of BU-OE in mice was 29.4 mg/kg (male) and 22.8 mg/kg (female), respectively. As for the long-term toxicity, BU-OE showed no apparent toxic effects except minor cardiotoxic effects which were reversible. In conclusion, submicron emulsion is a suitable delivery system for oral administration of bufadienolides, with satisfactory stability, superior antitumor efficacy and low toxicity.