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Congenital rubella syndrome is a global health problem. The incidence is much higher in Africa and Southeast Asia than the rest of the world, especially in countries where universal rubella vaccination has not been implemented. Healthcare worker’s knowledge on rubella infection and the rubella vaccine is of utmost importance in achieving and maintaining vaccination coverage targets. This study aimed to assess health care workers knowledge on rubella infection in Kilimanjaro Tanzania, after the introduction of a rubella vaccination. This was a health facility-based cross sectional study. It was conducted in three districts of the Kilimanjaro region between August and October 2016. The study involved eligible health care workers in selected health facilities. An interview guide was used for collecting information by face-to-face interviews. Multivariate analysis was used to assess factors associated with rubella knowledge among healthcare workers. A total of 126 health care workers were interviewed. An acceptable level of knowledge was considered if all five questions about rubella were correctly answered. Only 26.4% (n = 31) answered all questions correctly. In multivariate analysis education level and working department were predictors of rubella knowledge; health care workers with an advanced diploma had an adjusted odds ratio (AOR) of 7.7 (95% Confidence interval; CI: 1.4, 41.0), those with a university degree (AOR: 10; 95% CI: 2.4; 42.5) and health care workers in the outpatient department (AOR: 0.06; 95% CI: 0.04; 0.29). Our study confirmed that health care worker’s knowledge on rubella …