To determine the knowledge regarding hepatitis B virus (HBV) mother-to-child transmission (MTCT) and its prevention and treatment among healthcare workers (HCWs) in Guangdong Province, China, an HBV endemic area. An HBV knowledge questionnaire was administered to 900 HCWs from the 3rd Affiliated Hospital of Sun Yat-Sen University and 2 rural hospitals in Guangdong Province. The 27 items in the questionnaire fell into 3 sections: HBV MTCT general knowledge, respondents' practices of preventing HBV MTCT and awareness of the resources of preventing HBV MTCT. The data collected were coded and analysed using SPSS software version 20. In total, 503 of 900 HCWs responded to the survey (response rate: 55.9%). Eighty-four individuals responded correctly to all of the knowledge questions: 58 were doctors, and 26 were nurses (P < .05). Doctors more often performed practices than nurses (t = 3.591, P < .01). Participants from the infectious disease department demonstrated a significantly higher proportion of correct answers and resource utilization than other specialties (χ = 14.052, 7.998, P < .01). In terms of the average knowledge score, t test or ANOVA showed that there were significant differences between the specialty groups (t = 3.110, P < .01), hospital level groups (t = 2.337, P < .05) and age groups (F = 3.020, P < .05). Respondents' initiative increased with hospital level and age (t = 2.993, 7.493, P < .01). A considerable percentage of HCWs has misconceptions about HBV MTCT. Healthcare workers, in particular nurses, those working in noninfectious disease departments or township hospitals and younger medical staff, lack systematic and comprehensive knowledge about HBV MTCT and are in urgent need of HBV-related training.