Health care workers (HCWs) have high infection risk owing to treating patients with coronavirus disease 2019 (COVID-19). However, research on their infection risk and clinical characteristics is limited. To explore infection risk and clinical characteristics of HCWs with COVID-19 and to discuss possible prevention measures. This single-center case series included 9684 HCWs in Tongji Hospital, Wuhan, China. Data were collected from January 1 to February 9, 2020. Confirmed COVID-19. Exposure, epidemiological, and demographic information was collected by a structured questionnaire. Clinical, laboratory, and radiologic information was collected from electronic medical records. A total of 335 medical staff were randomly sampled to estimate the prevalence of subclinical infection among a high-risk, asymptomatic population. Samples from surfaces in health care settings were also collected. Overall, 110 of 9684 HCWs in Tongji Hospital tested positive for COVID-19, with an infection rate of 1.1%. Of them, 70 (71.8%) were women, and they had a median (interquartile range) age of 36.5 (30.0-47.0) years. Seventeen (15.5%) worked in fever clinics or wards, indicating an infection rate of 0.5% (17 of 3110) among first-line HCWs. A total of 93 of 6574 non-first-line HCWs (1.4%) were infected. Non-first-line nurses younger than 45 years were more likely to be infected compared with first-line physicians aged 45 years or older (incident rate ratio, 16.1; 95% CI, 7.1-36.3; P < .001). The prevalence of subclinical infection was 0.74% (1 of 135) among asymptomatic first-line HCWs and 1.0% (2 of 200) among non-first-line HCWs. No environmental surfaces tested positive. Overall, 93 of 110 HCWs (84.5%) with COVID-19 had nonsevere disease, while 1 (0.9%) died. The 5 most common symptoms were fever (67 [60.9%]), myalgia or fatigue (66 [60.0%]), cough (62 [56.4%]), sore throat (55 [50.0%]), and muscle ache (50 [45.5%]). Contact with indexed patients (65 [59.1%]) and colleagues with infection (12 [10.9%]) as well as community-acquired infection (14 [12.7%]) were the main routes of exposure for HCWs. In this case series, most infections among HCWs occurred during the early stage of disease outbreak. That non-first-line HCWs had a higher infection rate than first-line HCWs differed from observation of previous viral disease epidemics. Rapid identification of staff with potential infection and routine screening among asymptomatic staff could help protect HCWs.