Vaginal self-sampling for human papillomavirus (HPV) testing can potentially increase cervical screening coverage. This study aimed to investigate the acceptability of vaginal self-sampling for HPV testing and factors that might influence a woman's preference for this as a cervical screening method. This was a cross-sectional study that recruited 725 women from the urban and suburban areas of Selangor, Malaysia. All study participants were instructed to self-collect vaginal sample using a dry flocked swab before responding to a detailed questionnaire documenting their experience and preference for self-sampling. Most of the study participants (>80%) perceived vaginal self-sampling as easy, convenient, not embarrassing, comfortable, and were confident in performing the test. This suggests high acceptability toward vaginal self-sampling for HPV testing. Of the 725 women, 83% preferred self-sampling HPV testing over healthcare personnel sampling HPV testing and Pap test. Women with higher household income and full-time employment status were more likely to prefer self-sampling. Those who had not undergone Pap test also expressed preference for self-sampling HPV testing. Convenience and women's confidence in performing a vaginal self-sampling for HPV testing were the independent key factors that influenced the preference for self-sampling method. Vaginal self-sampling for HPV testing is highly acceptable among Malaysian women. It is the preferred choice as a primary cervical screening method and serves as an alternative to healthcare-acquired sample for Pap test. PREVENTION RELEVANCE: Organized cervical cancer screening remains unachievable in many countries. Self-sampling HPV testing is an evidence-based method that can remove barriers to cervical screening. This is particularly important for developing countries in order to achieve the WHO global strategy to accelerate cervical cancer elimination.