This study explores the changes in perceptions of transitional challenges faced by learners studying Chemistry through English-medium instruction (EMI) at a university in Japan over one academic semester. The research investigates the relationship between students’ English proficiency, academic performance, and their perception of transitional challenges. The study involves 27 students from an introductory Chemistry course and adopts a mixed-methods approach. The quantitative analysis utilises IELTS scores, as well as pre-and post-content tests, to examine the relationship between language proficiency and academic performance. Complementing these findings, the qualitative analysis provides insights from semi-structured interviews conducted at the start and end of a 12-week semester. These interviews offer an in-depth look into the individual transition processes of six purposefully selected students who represent a range of backgrounds and varying degrees of success in EMI, including both international and local students. The findings indicate that English proficiency alone does not guarantee a successful transition into EMI. Other factors play crucial roles in the rate and extent to which students overcome transitional challenges. The study recognises the need for ongoing support to help students navigate persistent transitional challenges in EMI. Implications are discussed regarding language support, EMI curriculum planning, and future research directions.