Background: In 2019, there was an outbreak of hepatitis A virus (HAV) infections in Korea. This study was performed to determine the seroprevalence of HAV among healthcare workers (HCWs) in a university-affiliated hospital and to establish an appropriate vaccination strategy against HAV for HCWs. Methods: Total antibody titers to HAV were measured using an electrochemiluminescence immunoassay kit in 1,466 HCWs. The seroprevalence of HAV and HAV vaccination rates in HCWs who were negative for anti-HAV were determined and compared among age and occupational groups. Results: In the whole study population, the seroprevalence of HAV was 49.9%. The seroprevalence was 56.3% in HCWs aged 20-24 years, decreased to 20.9% in those aged 35-39 years, and increased to 100% in those aged 60 years or more. Among HCWs who were seronegative for HAV, 70.6% received HAV vaccination. Among the occupational groups, the vaccination rate was the highest in the nurse group (80.6%) and the lowest in the doctor group (40.2%) (P<0.001). Conclusion: HCWs in their thirties and forties, who demonstrated the lowest seroprevalence of HAV among the age groups, should be prioritized for HAV vaccination. In addition to serologic tests for HAV, interventions to increase vaccination rates should be applied for HCWs, particularly doctors.