The aim of this study to evaluate knowledge, attitude and practice of rubber dam use among Sudanese dentists. Descriptive cross- sectional study among 250 Sudanese dentists (143 general dental practitioner and 57 specialist) working within Khartoum city. Self administered questionnaire including questions about knowledge, attitude and practice of rubber dam use. Years of experience, gender, speciality and place of graduation were compared and analysed by chi-square test with the level of significance set at p ≤ 0.05. The response rate was 80%. Rubber dam as a routine use practiced by only 4.5%, however 31% used it occasionally. Cotton roll and saliva ejector were the methods of isolation for 40.5% of those doing dental operative procedure. Almost majority (69%); said they received training in rubber dam use during undergraduate period. The most barriers against rubber dam usage, was unavailability (45.5%). In spite of the majority of Sudanese dentists received training on rubber dam use during under graduate, few practiced it regularly and the main barrier was unavailability.