Background: Enterocytozoon bieneusi is the most common microsporidian species that can infect humans and various animals worldwide. To date, there has been limited information on the prevalence and genotypes of E. bieneusi infection in cattle in the Republic of Korea. Therefore, this study investigated the prevalence and genotypes of E. bieneusi circulating in pre-weaned Korean native calves and determined the age pattern of E. bieneusi infection and the relationship between E. bieneusi infection and diarrhea. Methods: The prevalence of E. bieneusi infection in pre-weaned Korean native calves was screened by polymerase chain reaction. PCR-positive products were sequenced to determine the genotype of E. bieneusi . A Chi-square analysis was used to compare the association between diarrhea and the infection rate of E. bieneusi in each age ranges or all ages. Results: PCR and sequencing analysis revealed an overall prevalence of E. bieneusi of 16.9% (53/314) in pre-weaned calves. The prevalence of E. bieneusi was highest in September (36.2%), followed by March (28.3%). E. bieneusi infection (χ 2 = 5.82, P = 0.016) was associated with diarrhea in calves. Our results also indicated that E. bieneusi infection was statistically associated with calf age (χ 2 = 11.61, P = 0.003), and the prevalence of E. bieneusi infection was significantly higher in calves aged 21-40 days (odds ratio = 2.90, 95% confidence interval: 1.54-5.45; P = 0.001) than in those aged 1-20 days. Interestingly, the association between E. bieneusi infection and diarrhea was observed only in calves aged 1-20 days (χ 2 = 5.82, P = 0.010). Furthermore, our results indicated that E. bieneusi infection in pre-weaned calves may be associated with mild diarrhea rather than severe diarrhea. We identified three known genotypes, BEB4 (n = 12), BEB8 (n = 21), and J (n = 16), and three novel genotypes, BEB8-like (n = 21), KCALF1 (n = 1), and KCALF2 (n = 1); all of which belonged to Group 2. The genotype BEB8 was the most prevalent among all age groups, irrespective of diarrhea. All genotypes identified in this study exhibited zoonotic potential. Conclusions: To our knowledge, this is the first report of the presence of the genotype BEB4 in pre-weaned Korean native calves. Zoonotic E. bieneusi infection was prevalent in pre-weaned Korean native calves, indicating that cattle may play an important role as a reservoir host for E. bieneusi transmission to humans.