A 12.4-kDa peptide, corresponding to the entire ORF3 protein of hepatitis E virus (HEV), derived from human HEV genotype 4 and expressed in Escherichia coli as a fusion protein with a 17.5-kDa fragment of interleukin (IL)-1beta at the N-terminus, was recognized by HEV-reactive sera. Eight monkeys were immunized with the purified peptide, and seven were used as non-immunized controls. All 15 monkeys were challenged with HEV genotype 1 or 4. All control animals developed infection and hepatitis, and all but one vaccinated monkey became infected. Nevertheless, the vaccine was effective in reducing the virus titer and shortening the duration of viremia and fecal shedding. Furthermore, the vaccine provided some protection against hepatitis (1 of 2 monkeys in the two-dose regimen and 4 of 6 in the three-dose regimen did not develop severe hepatitis) compared to the controls. These results suggest that immunization with the bacterially expressed peptide may partially prevent experimental hepatitis, and even infection, in primates, following intravenous challenge with high doses of two HEV genotypes.