Separated cycling infrastructure, together with other investments such as bike share systems, are increasingly seen as important tools for planners and policy makers to promote urban cycling and multi-modal transportation systems. However, determining causality – that the construction of separated cycling infrastructure promotes higher levels of ridership – presents a significant challenge to researchers. This study uses 495,084 Hamilton Bike Share (HBS) trip routes, derived from GPS trajectories and a custom map-matching algorithm, to inform a longitudinal analysis concerning the influence of 10 separated cycling infrastructure investments made by the City of Hamilton between January 2019 and March 2022. Monthly bicycle kilometers traveled (BKT) along the segments, to account for variability in level of segment use per trip, was modeled using fixed effects regression, which controls for time and location-specific effects, climatological, and contextual factors. A suite of variables indicating completion of the various interventions were included, and of the 10 interventions, five were found to have highly positive impacts (71.60% to 441.95% increases) on monthly BKT. Heterogeneity across the investments was explored. Concrete-barrier separated cycle tracks were the most common interventions with significant and positive coefficients in the model, as were segments more connected with other separated cycling infrastructure, and more accessible to system ridership. This study provides an empirical, longitudinal, city-wide examination of the effects of cycling infrastructure on bike share ridership, setting a framework for further study of private cycling and the nature of the increases in bike share traffic across the interventions of interest.