Care of newborn is traditionally provided by mothers. Maternal knowledge and practice of newborn care can significantly affect neonatal morbidity and mortality. There is convincing concern to explore knowledge and practice of postnatal mothers regarding newborn care. The objective of the study was to evaluate knowledge and practice of postnatal mothers regarding newborn care at hospital setting. This cross-sectional observational study was carried out in Special Care Newborn Unit, Rangpur Medical College Hospital, Bangladesh from July 2018 to January 2019. In this study, mean maternal age was 23.27±3.87 years. Out of 150 respondent mothers 121(81.0%) possessed average knowledge and 144(96.0%) demonstrated average practice on newborn care. Level of knowledge was high in the area of clean cord care by using sterile thread 125(83.3%), p<0.001, duration of exclusive breastfeeding 100(66.7%), p<0.001), colostrum as first food of newborn 122(81.3%), p<0.001) and awareness about immunization 95(63.3%), p<0.001). Postnatal mothers demonstrated poor knowledge regarding cord stump stuff 41(27.3%) p<0.001) and first bath of newborn 38(25.3%), p<0.001. Mothers showed higher level of practice in the area of wash and clean napkins after motion 147(98.0%), p<0.001 and poor practice in correct position and attachment of breastfeeding 00(00.0%), p=0.001. Most postnatal mothers practiced exclusive breastfeeding 139(92.7%), p<0.001. Association between knowledge and practice of newborn care was non significant (p=0.305). Results of the present study demonstrated that majority of the mothers had average knowledge and practice regarding newborn care. Level of knowledge was high in clean cord care using sterile thread, colostrum as first food, duration of exclusive breastfeeding and awareness about immunization. Mothers had poor knowledge about cord stump material and first bath of newborn. Mothers claimed higher level of practice in the area of napkin wash and clean after motion, eye care, exclusive breastfeeding and poor practice in correct position and attachment of breastfeeding.