Objectives This study aimed to investigate differences in the anti-hepatitis A virus (HAV) antibody seropositivity rate by age and gender. Methods: We collected information on anti-HAV immunoglobulin G and immunoglobulin M status from samples submitted for HAV antibody testing in 2012–2022. A total of 1,333,615 cases were included in the analysis. Results: By age, the seropositivity rate was represented by a U-shaped curve, such that the rate was low for the group aged 20 to 39 years and higher in those who were younger or older. Over time, the curve shifted rightward, and the seropositivity rate declined gradually in the group aged 35 to 39 years and older. A gender-based difference in antibody seropositivity rate was especially noticeable in the group aged 20 to 29 years. This difference between genders widened in the participants’ early 20s—when men in the Republic of Korea enlist in the military—and the divergence continued subsequently for older individuals. Conclusion: These results indicate a higher risk of severe infection among older individuals and a gender-based difference in seroprevalence. Therefore, it is necessary to implement policies to promote vaccination in adults.