INTRODUCTION: Walking and cycling bring health and environmental benefits, but there is little robust evidence that changing the built environment promotes these activities in populations. This study evaluated the effects of new transport infrastructure on active commuting and physical activity. STUDY DESIGN: Quasi-experimental analysis nested within a cohort study. SETTING/PARTICIPANTS: Four hundred and sixty-nine adult commuters, recruited through a predominantly workplace-based strategy, who lived within 30 kilometers of Cambridge, United Kingdom and worked in areas of the city to be served by the new transport infrastructure. INTERVENTION: The Cambridgeshire Guided Busway opened in 2011 and comprised a new bus network and a traffic-free walking and cycling route. Exposure to the intervention was defined using the shortest distance from each participant's home to the busway. MAIN OUTCOME MEASURES: Change in weekly time spent in active commuting between 2009 and 2012, measured by validated 7-day recall instrument. Secondary outcomes were changes in total weekly time spent walking and cycling and in recreational and overall physical activity, measured using the validated Recent Physical Activity Questionnaire. Data were analyzed in 2014. RESULTS: In multivariable multinomial regression models--adjusted for potential sociodemographic, geographic, health, and workplace confounders; baseline active commuting; and home or work relocation-exposure to the busway was associated with a significantly greater likelihood of an increase in weekly cycle commuting time (relative risk ratio=1.34, 95% CI=1.03, 1.76) and with an increase in overall time spent in active commuting among the least active commuters at baseline (relative risk ratio=1.76, 95% CI=1.16, 2.67). The study found no evidence of changes in recreational or overall physical activity. CONCLUSIONS: Providing new sustainable transport infrastructure was effective in promoting an increase in active commuting. These findings provide new evidence to support reconfiguring transport systems as part of public health improvement strategies.