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Background: Cycling is beneficial for health and the environment but the evidence on the overall and differential impacts of interventions to promote cycling is limited. Here we assess the equity impacts of funding awarded to support cycling in 18 urban areas between 2005 and 2011. Methods: We used longitudinally linked 2001 and 2011 census data from 25,747 individuals in the Office for National Statistics Longitudinal Study of England and Wales. Logistic regression was used to assess the impacts of funding on commute mode as the interaction between time and area (intervention/comparison) in individual-level difference-in-difference analyses, adjusting for a range of potential confounding factors. Differential impacts were examined by age, gender, education and area-level deprivation, and uptake and maintenance of cycling were examined separately. Results: Difference-in-difference analyses showed no intervention impact on cycle commuting prevalence in the whole sample (AOR = 1.08; 95% CI 0.92, 1.26) or among men (AOR = 0.91; 95% CI 0.76, 1.10) but found an intervention effect among women (AOR = 1.56; 95% CI 1.16, 2.10). The intervention promoted uptake of cycling commuting in women (AOR = 2.13; 95% CI 1.56, 2.91) but not men (AOR = 1.19; 95% CI 0.93, 1.51). Differences in intervention effects by age, education and area-level deprivation were less consistent and more modest in magnitude. Conclusions: Living in an intervention area was associated with greater uptake of cycle commuting among women but not men. Potential gender differences in the determinants of transport mode choice should be considered in the design and evaluation of future interventions to promote cycling.