Purpose Rail level crossing removals to improve transport performance across metropolitan Melbourne (state of Victoria) resulted in new rail fencing and grade-separation of tracks from the surrounding environment at several sites. These design changes restricted pedestrian access to the rail tracks, which is a countermeasure known to prevent railway suicide in other settings. We examined whether any such suicide prevention effect followed the removals. Methods We used a multiple-arm pre-post design to test whether a decrease in monthly frequency of railway suicides occurred at level crossing removal sites (intervention sites), compared to randomly matched sites where level crossings had not yet been removed (control sites). We used data available in the Victorian Suicide Register covering the period 1st January 2008 to 30th June 2021. Results The mean monthly number of railway suicides decreased by 68% within a 500 m radius of intervention sites (RR: 0.32; CI 95% 0.11–0.74) and by 61% within a 1000 m radius of intervention sites (RR: 0.39; CI 95% 0.21–0.68). There was no evidence that the mean monthly number of railway suicides changed at the control sites, either within a 500 m radius (RR: 0.88; CI 95% 0.47–1.56) or a 1000 m radius (RR: 0.82; CI 95% 0.52–1.26). Conclusion The reduction in railway suicides at locations where level crossings were removed, demonstrates the suicide prevention benefits that can be derived from a major infrastructure project even if not initially intended. Planning for major infrastructure projects should include consideration of these benefits, with designs incorporating features to maximise suicide prevention impact.