This paper reports on the relationship between the pre-service teachers’ enacted Topic Specific Pedagogical Content Knowledge (eTSPCK) observable in the classroom teaching of Organic Chemistry and learner achievement in the topic. The study employed a mixed-methods research design with a sample of 17 pre-service teachers (PSTs). The PSTs were drawn from a Chemistry methodology class exposed to an intervention which focused on the pedagogical transformation of content knowledge of Organic Chemistry at a South African University. A sub-set of 4 of the PSTs was followed during a teaching practicum where the PSTs conducted lessons on Organic Chemistry with 88 Grade 12 Secondary School physical sciences learners in South Africa. Data collected were the video-recordings of the 4 PSTs’ classroom teachings, lesson plans, pre-and post-lesson interviews, and learners’ content knowledge pre-and post-achievement tests on Organic Chemistry prior to and immediately after the PSTs’ teachings, respectively. The video recorded lessons were analysed using an in-depth qualitative method of identifying TSPCK episodes and the interviews were analysed using content analysis. The learners’ achievement tests were scored using a memorandum of correct responses. The Rasch Model Analysis, Analysis of Covariance and Correlation analysis were employed in determining the relationship between PSTs’ eTSPCK and learner achievement. The analysis revealed a statistically significant difference across the means of the learners’ pre/post-tests, as well as a significant positive correlation between pre-service teachers’ eTSPCK and learners’ post-test achievement following the teaching. The correlation was found to increase in positive strength with the displayed higher quality categories of TSPCK episodes. The implications and recommendations for science teacher education are discussed.