Background: Pre Exposure Prophylaxis and Post exposure ART prophylaxis is considered the standard preventive measure against HIV/AIDS infection among the exposed. Several clinical studies have demonstrated that HIV transmission can be reduced by 81% following the immediate administration of antiretroviral agents. With number of interventions undertaken to scale up uptake the ART prophylaxis (pre and post exposure prophylaxis) in line with behaviour change programs especially in high risk population in Tanzania, little is known about knowledge, attitude and ART prophylaxis uptake among University Students in Kilimanjaro Region. Objectives: To determine prevalence, knowledge and attitude of ART prophylaxis uptake among University students in Kilimanjaro region. Methods: A descriptive cross sectional study design was conducted from May to June 2016 among University students in Kilimanjaro region. 324 university students were included in this study. A self-administered questionnaires was used to collect information. SPSS version 20 was used for data analysis. Categorical data were summarised in to frequency and percentages. Results are presented in tables. Results: A total of 324 University students participated in this study of these 21.3% exposed to blood, body fluids, needles or sharp objects. Prevalence of ART prophylaxis uptake among those exposed was 36.2%. About two third of all respondents (65.7%), were reported to have inadequate knowledge about post exposure prophylaxis for HIV. The majority of respondent (85%) had good attitude toward the ART prophylaxis. Conclusion: This study found that the prevalence of ART prophylaxis uptake among University students to be low compared to other studies, this might be due to their inadequate knowledge on ART prophylaxis and HIV risky situations. Even though many of the students had HIV risky exposure, the reported proportion of university students that were exposed and did not take the ART prophylaxis for HIV cannot be considered low. There is also low knowledge on ART prophylaxis despite the positive attitude among university students in Kilimanjaro region. This study recommends enforcement of HIV/AIDS policy and ART guideline in universities and awareness programs (like seminars) as well as counselling services.