Background: Human papillomavirus (HPV) has been described as an ‘equal opportunity’ pathogen affecting both sexes and the leading cause of cervical cancer. Vaccination against oncogenic HPV types is a significant step towards decreasing the prevalence, morbidity, and mortality associated with cervical cancer. Objectives: To investigate attitude and perceived barriers to uptake of HPV vaccine among female students in a Nigerian Tertiary Institution. Methods: A cross-sectional questionnaire-based study that was conducted in the University of Nigeria, Enugu Campus from March 01 to April 01, 2017. The representative sample size of 320 female undergraduates was selected using a multistage sampling technique. Data were entered using Microsoft Excel Windows 7 and exported to IBM SPSS version 20.0 software for analysis. Results: Total 38 (13.0%) participants had received HPV vaccine. Majority of the participants had a positive attitude towards HPV vaccination (2.5 ± 6.4). The major perceived barriers to uptake of vaccination were: exorbitant cost of the vaccine (94.1%) and lack of time due to lectures (53.0%). Age of the participants had no influence on the uptake of HPV vaccine whereas department, ethnic group, and marital status had a strong influence on vaccination uptake (p<0.05). Conclusion: A low proportion of the participants had received the HPV vaccine. Majority of the participants had a positive attitude towards HPV vaccination. The major perceived barrier to vaccination was the exorbitant cost of the vaccine. The university in collaboration with the Ministry of Health should design strategies to enlighten the university community on HPV infection through seminars and workshops.