Background: Occupational accidents contribute 46-65% of hepatitis B virus (HBV) infections among healthcare workers (HCWs). Vaccine against HBV infection offers protection of more than 90%. Despite availability of the vaccines in Tanzania, it’s uptake among HCWs in primary health facilities remains understudied. This study explored HBV vaccine uptake and associated factors among HCWs in rural and urban settings of north western Tanzania. Methodology: A cross-sectional analytical study was conducted between June and July in 2022 among HCW in Misungwi and Ilemela districts. Data were collected using a self-administered questionnaire and analyzed using IBM SPSS® version 25. Results: A total of 402 HCWs were recruited, their mean age was 34.9±7.77 years. Approximately half (54.7% (220/402) of HCWs had received at least one shot of HBV vaccine with only (18% (76/402) being fully vaccinated. Ilemela showed significantly higher uptake (χ2=23.64, df=1, p=0.00) of HBV vaccine as compared to their counterparts in Misungwi. Being male (aOR=2.38, 95% CI 1.28-4.45, p=0.006), working in urban (aOR=5.75, 95% CI 2.91-11.35, p=0.00) and employment duration of more than two years (aOR=3.58, 95%CI 1.19-10.74, p=0.023) were significantly associated with higher odds of HBV vaccination. Moreover, high perceived susceptibility to HBV infection (aOR=2.20, 95% CI1.02-4.75, p=0.044) and history of needle prick injuries (aOR=6.87, 95%CI 3.55-13.26, p=0.00) were also significantly associated with higher odds of HBV vaccination. Conclusion: There was low uptake of HBV vaccine among HCW in primary health facilities with a noteworthy difference between rural and urban settings.. Therefore, advocacy campaigns as well as resource mobilization towards promotion of HBV vaccination in primary health facilities are pivotal.