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Hepatitis E virus (HEV) infection is a major public health concern in low-income countries, yet incidence and prevalence estimates are often lacking. Serum (<i>n</i> = 653) and faecal (<i>n</i> = 150) samples were collected from apparently healthy individuals using convenience sampling technique in six communities (Ore, Oke-Osun, Osogbo, Ede, Esa-Odo, and Iperindo) from Osun State, Nigeria. Serum samples were analysed for total anti-HEV IgG/IgM and anti-HEV IgM using commercially available HEV ELISA kits. Total anti-HEV positive serum and all stool samples were analysed for HEV RNA by RT-PCR. Overall, 15.0% (<i>n</i> = 98/653) and 3.8% (<i>n</i> = 25/653) of the serum samples were positive for anti-HEV total and IgM antibodies, respectively. Total anti-HEV and IgM in Ore, Oke-Osun, Osogbo, Ede, Esa-Odo, and Iperindo was 21.0% (<i>n</i> = 13/62) and 3.2% (<i>n</i> = 2/62), 19.4% (<i>n</i> = 20/103) and 6.8% (<i>n</i> = 7/103), 11.4% (<i>n</i> = 12/105) and 2.9% (<i>n</i> = 3/105), 8.0% (<i>n</i> = 16/199) and 1.5% (<i>n</i> = 3/199), 22.0% (<i>n</i> = 22/100) and 10.0% (<i>n</i> = 10/100), and 17.9% (<i>n</i> = 15/84) and 0.0% (<i>n</i> = 0/84), respectively. All samples (stool and serum) were HEV RNA negative. Anti-HEV seroprevalence was associated with rural location, increasing age, alcohol consumption, and rearing of animals. This study demonstrated a high anti-HEV seroprevalence in Osun State, indicating the need to implement surveillance and asses the hepatitis E burden in Nigeria.