Introduction: The practice of Pap smear has long been an issue worldwide especially in the developing countries. Malaysia National Health and Morbidity Survey IV reported an uptake of 12.8% in 2011. It was found that Malay women has the lowest prevalence of uptake compared to other major ethnicities in the country. The objective of this study is to determine the prevalence of Pap smear practice and its predictors among staff in a public university with majority of Malay ethnicity. Methodology: A cross-sectional study was conducted among female staff in a public university in Nilai, Negeri Sembilan using a self-administered and validated questionnaire. All female staff from the seven faculties and the library were invited to participate. Data were analysed using SPSS version 22 for descriptive and inferential statistics, whereby Multiple Logistic Regression were performed to identify the predictors. Results: One-hundred-and eighty-seven respondents participated this study. The prevalence of Pap smear practice was 65.2%, of which, two-thirds (65.6%) had done it within the past three years. Predictors of the practice were higher parity, attending annual medical check-up, husband’s support and positive attitude towards the test. Conclusion: The prevalence of Pap smear uptake among this population is higher than other studies in Malaysia, however, not all of them have done it regularly within 3 yearly interval as suggested by the World Health Organisation.