In the growing body of English medium instruction (EMI) research, few studies have directly compared the effects of medium of instruction on subject learning. This study compares direct measures of content knowledge and perceptions of knowledge acquisition for students studying Chemistry via English (n = 27) and Japanese Medium Instruction (JMI) (n = 26). Data were collected at a university in Japan where Chemistry courses were taught in both Japanese and English as part of a parallel program offering the same undergraduate curriculum in either of the two languages of instruction. An analysis was undertaken of students' learning outcomes measured by pre-post course content tests. These measurable test outcomes were triangulated with data from student interviews (n = 17) to identify differences in the learners' perceived experiences according to the medium of instruction. While the quantitative results revealed no significant overall differences in the adjusted post-test scores between EMI and JMI students, the qualitative data offered more detailed insight into the participants' perspectives of content learning, highlighting unique challenges faced only by the EMI group. Findings point to implications for educational provision in contexts where the global trend of EMI has largely been unaccompanied with research evidence on its cost-effectiveness.