Professional development has been identified as an effective way to increase college STEM instructors’ use of research-based instructional strategies (RBIS) known to benefit student learning and persistence in STEM. Yet only a few studies relate professional development experiences to later teaching behaviors of higher education instructors. This study of 361 undergraduate mathematics instructors, all of whom participated in multi-day, discipline-based workshops on teaching held in 2010–2019, examined the relationship between such participation and later use of RBIS. We found that instructors’ RBIS attitudes, knowledge, and skills strengthened after participating in professional development, and their self-reported use of RBIS became more frequent in the first year after the workshop. Applying the Theory of Planned Behavior as a conceptual framework, we used a structural equation model to test whether this theory could explain the roles of workshop participation and other personal, professional and contextual factors in fostering RBIS use. Findings indicated that, along with workshop participation, prior RBIS experience, class size, and course coordination affected RBIS use. That is, both targeted professional development and elements of the local context for implementation were important in supporting instructors’ uptake of RBIS—but, remarkably, both immediate and longer-term outcomes of professional development did not depend on other individual or institutional characteristics. In this study, the large sample size, longitudinal measurement approach, and consistency of the form and quality of professional development make it possible to distinguish the importance of multiple possible influences on instructors’ uptake of RBIS. We discuss implications for professional development and for institutional structures that support instructors as they apply what they learned, and we offer suggestions for the use of theory in future research on this topic.