Hepatitis B vaccine (HepB) is an effective tool in prevention of hepatitis B virus (HBV) infection. When administered at birth, it prevents mother-to-child transmission of acute and chronic HBV infection. However, despite a decade and half of implementation of HepB birth dose (HepB-BD), uptake has remained persistently low in Enugu State, Nigeria. We assessed the uptake of valid HepB-BD and the reasons given by mothers of infants for not receiving the HepB-BD in Enugu State, South-east Nigeria.An hospital-based cross-sectional survey was conducted among mother-infant pairs attending immunization clinics at randomly selected health facilities in Enugu State, Nigeria. Overall, 344 mothers and their infant children in this study were interviewed using structured questionnaire. Data on maternal reasons for non-receipt of valid HepB-BD by their infants and their recommendations on ways to improve valid HepB-BD uptake, were collected. We defined valid birth dose as the receipt of first dose of HepB within 24 h of birth.Overall, 254 (73.8%) infants did not receive valid HepB-BD. Major reasons for its non-receipt were vaccine not available at place of delivery (91.3%, n = 232), delivery did not take place on immunization day (75.6%, n = 192), lack of awareness on timing of valid HepB-BD (72.8%, n = 185), long distance from the health facility (5.1%, n = 13) and fee payment for immunization (6.3%, n = 16). Of the 384 maternal recommendations, 143 (37.2%) emphasized female literacy while 87 (22.7%) indicated pre-positioning the vaccines at labor rooms to improve valid HepB-BD uptake.The low receipt of valid HepB-BD among infants attending routine immunization clinics, found in this study were attributed to lack of maternal awareness on timing of HepB-BD and poor integration of child delivery and immunization services. We recommend educating mothers on benefits of a timely HepB-BD and pre-positioning the vaccines at the labor rooms.