Background HIV Self-Testing (HIVST) is universally accepted as an innovative strategy complimenting existing HIV testing services to archive the UNAIDS, 95-95-95 goals by 2030. However, the adoption of HIVST is lagging in most sub-Saharan countries, including Tanzania. This study aimed to determine the level of knowledge, acceptability, and willingness to use HIVST among Medical students in Kilimanjaro region, Tanzania. Methods A descriptive cross-sectional study using a self-administered, semi-structured questionnaire was conducted from May to June 2019 among 271 medical students aged 18–44 years enrolled in a degree of Medicine course at Kilimanjaro Christian Medical University College. Results A total of 271 participants were enrolled (response rate of 100%). The mean age was 23.9 (SD 2.9), the majority (91%) were Christians, being single (92%), and a half (50.2%) were males. More than half (55.7%) was sexually active, 67.5% reported the age of first sexual debut at 19 years and above. The majority (81.5%) reported that they had one sexual partner, 37% used condoms during the last sexual act. The majority (98.7%) had never had a sexually transmitted disease during the past 3 months, 22.5% reported using alcohol when having sex. More than three-quarters (79%) ever tested for HIV, and 41.6% tested for HIV in the past year. More than two-thirds (67.9%) had a high level of knowledge on oral fluid HIV self-test. Being a female was related with high level of knowledge (P = 0.225). The acceptability of HIVST was 62.7%, and about two-thirds showed a willingness to buy a self-test kit if available for public use. Conclusions The high level of knowledge on oral fluid HIV self-testing, acceptability and willingness to buy self-test kit if available for public use among sexually active medical students underscores the importance of introducing HIVST as a complementary approach for existing HIV testing services in this setting. To make HIVST effective, interventionist should address concerns associated with self-testing, such as lack of pre and post-test counseling, suicidal risks after receiving HIV positive results, perceived risks of inaccurate HIVST test results, lack of linkage to care of individuals receiving HIV positive results, perceived risks of intimate partner violence, coercive testing of a female partner, and perceived high cost of buying self-test kits.