Successful implementation of Universal Test and Treat as a strategy to achieve the 90-90-90 target requires higher HIV testing rates. Currently, uptake of HIV testing is not optimal which has directed research initiatives towards identification of additional HIV testing methods. HIV self-testing (HIVST) has received growing attention as a complementary testing approach as it overcomes barriers that are commonly associated with current HIV testing methods. In sub-Saharan Africa, acceptability rates showed a gendered pattern of men benefitting more than women, with limited evidence to explain this difference. This study assessed whether men or women in KwaZulu-Natal displayed a higher acceptance of HIVST and also explored factors that influenced and motivated their acceptability. Participants were recruited through purposive sampling at two clinical research sites to participate and underwent qualitative assessments. The outcomes from focus group discussions coupled with findings from a scoping review informed the design and data collection instruments for in-depth interviews. A randomised cross-over study design exposed participants to HIV counselling and testing and HIVST, accompanied by before (baseline) and after in-depth interviews. HIVST was acceptable among most participants with acceptability higher in women. Men preferred HIVST due to convenience and efficiency, whilst women favoured HIVST due to its potential to provide autonomy and empowerment. Also, lack of HIV counselling and managing a positive HIV result as well as linkage to care were raised as deterrents of HIVST. As HIVST was acceptable by most participants, future research efforts should be directed towards evaluating the feasibility of its introduction into the public health sector.