BACKGROUND: Cervical cancer is the second leading cause of death among women in Thailand. Understanding health literacy and knowledge of HPV, cervical cancer, and their screening is vital for nursing students as they prepare for professional practice. OBJECTIVES: To identify factors influencing health literacy and knowledge of HPV infection, cervical cancer, and screening among Thai nursing students at rural and urban universities. DESIGN: A cross-sectional descriptive comparative study. PARTICIPANTS/SETTINGS: Current undergraduate nursing students from rural and urban universities in Thailand. METHODS: A self-administered survey assessed health literacy and knowledge related to HPV infection, cervical cancer, and their screening. Descriptive and logistic regression analyses were used to identify factors associated with these variables. RESULTS: Among 641 nursing students (255 rural students and 386 urban students), rural students had significantly higher mean scores in cervical cancer screening knowledge (t = -4.86, p <
0.001) and health literacy compared to urban students (t = -2.57, p = 0.01). Rural students using the internet more than three days per week were 1.92 times more likely to have better knowledge of HPV and cervical cancer (95 % CI: 1.10-7.73). Awareness of HPV (ORa = 2.51, 95 % CI: 1.31-4.80) and frequent internet use (ORa = 2.31, 95 % CI: 1.04-5.12) were associated with higher knowledge about cervical cancer screening and health literacy among rural nursing students. CONCLUSIONS: Rural students exhibit higher health literacy and knowledge about cervical cancer screening than urban students. These findings highlight the need for diverse teaching strategies to enhance students' understanding and competency.