The goal of this study is to examine the role of knowledge and epistemological beliefs in the relationship between STEM (science, technology, engineering, and mathematics) professional development programme (SPDP) and instructional practices in a longitudinal study. To achieve this goal, it first determined the relationship and impact of SPDP on the implementation of instructional practices by comparing two groups (experimental and control group) of chemistry teachers after two years SPDP in Ekiti State, Southwestern, Nigeria. Data were collected from 90 teachers after the programme at two different measurement points through chemistry STEM-integrated knowledge assessment, questionnaires, and classroom observation checklists. Data were analyzed using t-test, bivariate correlation and structural equation modeling (SEM). Results indicated that teachers in the experimental group scored higher than their counterpart in the other group on all measures of instructional practices at both time intervals. However, there was no significant difference on overall instructional practices at time 1 but given the time interval, a significant difference exists. Teachers' participation in the SPDP was found to be highly correlated with STEM knowledge but weakly correlated with epistemological beliefs at time 1. Most importantly, STEM knowledge and epistemological beliefs mediated the relationship between SPDP and instructional practices at time 2. Implications for educational bodies, researchers, teachers and organizations planning to invest in teachers' SPDP as well as recommendations for future research directions are discussed.