Epsilon toxin (ETX) is secreted by Clostridium perfringens (C. perfringens)as a relatively inactive prototoxin (pETX), which is enzymatically activated to ETX by removing carboxy-terminal and amino-terminal peptides. Genetically engineered ETX mutants have been shown to function as potential vaccine candidates in the prevention of the enterotoxemia caused by C. perfringens. In the present study, two recombinant site-directed mutants of pETX, rpETX (rpETX) and rpETX (rpETX), were synthesized by mutating four essential amino acid residues (Tyr30, Tyr71, His106, Tyr196 or Phe199). Compared to recombinant pETX (rpETX), both rpETX and rpETX lacked the detectable toxicity in MDCK cells and mice, which suggested that both rpETX and rpETX are sufficiently safe to be vaccine candidates. Despite the fact that rpETX and rpETX were reactogenic with polyclonal antibodies against crude ETX, both single- and double-dose vaccination (V and V, respectively) of rpETX induced a higher level of IgG titer and protection in mice than that of rpETX. Therefore, we selected rpETX for the further study. Sheep received V of 150 μg rpETX developed significant levels of toxin-neutralizing antibodies persisting for at least 6 months, which conferred protection against crude ETX challenge without microscopic lesions. These data suggest that genetically detoxified rpETX could form the basis of a next-generation enterotoxemia vaccine.