Context: Road safety measures have been reported to effectively prevent or reduce the impact of road traffic injuries.Objectives: This study assessed private motorists’ knowledge and practice of road safety measures in Uyo, Nigeria.Methodology: This was a descriptive cross – sectional study. Data was collected using self administered semi- structured questionnaire and analyzed with SPSS version 20. Frequencies and associations were generated. Level of significance was set at 5%.Result: A total of 388 respondents participated in the study. The mean age of respondents was 37.67±8.27 years with male to female ratio of 1.75:1. Majority, 83.2% had tertiary education. Only 179 (46.1%) learnt how to drive in driving schools. The prevalence of road traffic injuries was 22.7%. Majority, 342 (88.1%) possessed driving licenses, out of which only 168 (49.1%) underwent driving tests. Level of knowledge of road signs ranged from 6.2% for “no stopping” to 67% for “no parking”. Lifetime consumption of alcohol was 55.7%. Eighty two (21.1%) of the respondents sometimes or never used seatbelts. The most common reason given by 65.9% was discomfort. Use of seat belt by back passengers was 15.7%. The average speed on highway was 106.9 ± 24.2 km/hour. Awareness of existence of speed limit in Nigeria was 63.7% and 156 (40.2%) admitted to making or receiving calls while driving. There was a statistically significant association between making or receiving calls and occurrence of road traffic injuries.Conclusion: Awareness campaigns to improve knowledge of highway codes and other traffic regulations are advocated for this study population and other private motorists in the study area.