The purpose of this study was to determine cross-protection between HEV genotypes 1 and 4, which are prevalent in China. Fecal suspensions of genotypes 1 and 4 from patients, as well as genotype 4 from swine, were inoculated intravenously into rhesus macaques. Each inoculum contained 5 × 104 genome equivalents of HEV. After infection, serum and fecal samples were collected serially and the levels of alanine aminotransferase (ALT) and anti-HEV IgG and IgM in sera, and HEV RNA in fecal samples, were measured. Liver biopsies were carried out. All the infected monkeys (12/12) developed anti-HEV IgG and exhibited fecal shedding of virus. IgM was detected in 11 of 12, and ALT elevation occurred about 2–6 weeks post-inoculation in 10 of 12, infected monkeys. Hepatic histopathology was consistent with acute viral hepatitis and the ORF2 antigen of HEV was detected in the granular cytoplasm of hepatocytes by immunohistochemistry. After recovery from their initial HEV infection, the monkeys were challenged with a heterologous genotype or heterologous source of HEV and monitored for hepatitis and fecal shedding. Previous infection with HEV completely or partially protected against subsequent challenge with a heterologous virus, because 7 of 11 monkeys did not develop HEV infection or shed virus in the feces, and none of them developed hepatitis or exhibited ALT elevation or liver biopsy findings of hepatitis. In conclusion, previous HEV infection may give rise to cross-genotype and cross-host-species protection. J. Med. Virol. 80:824–832, 2008. © 2008 Wiley-Liss, Inc.