This study explores how gender and first language (L1) conceptual transfer influence the use of English prepositions among 50 Vietnamese university students aged 18 to 25, enrolled in an English language program. The participants' comprehension of English prepositions in terms of usage, meaning, and context was assessed using pre- and post-tests consisting of 20 multiple-choice questions. The data analysis employed descriptive and inferential statistics, including chi-square tests and independent samples t-tests. Both the pre- and post-test results revealed a moderate level of English prepositional usage, with all participants displaying improvement. Female participants performed slightly better than males on the post-test, while there were no notable gender differences in pre-test scores. In both tests, participants with high levels of L1 conceptual transfer performed significantly poorer compared to those with low levels. These findings highlight the significant role of L1 conceptual transfer in the English prepositional usage of Vietnamese language learners, while suggesting a minor impact of gender. Further research is needed to delve deeper into this relationship. The results emphasize the importance of addressing L1 conceptual transfer in English language instruction, specifically regarding prepositions, within the context of Vietnam.