Introduction Active commuting-walking and bicycling for travel to and/or from work or educational addresses-may facilitate daily, routine physical activity. Several studies have investigated the relationship between active commuting and commuting stress; however, there are no studies examining the relationship between solely bicycle commuting and perceived stress, or studies that account for environmental determinants of bicycle commuting and stress. The current study evaluated the relationship between bicycle commuting, among working or studying adults in a dense urban setting, and perceived stress. Methods A cross-sectional study was performed with 788 adults who regularly travelled to work or study locations (excluding those who only commuted on foot) in Barcelona, Spain. Participants responded to a comprehensive telephone survey concerning their travel behaviour from June 2011 through to May 2012. Participants were categorised as either bicycle commuters or non-bicycle commuters, and (based on the Perceived Stress Scale, PSS-4) as either stressed or non-stressed. Multivariate Poisson regression with robust variance models of stress status based on exposures with bicycle commuting were estimated and adjusted for potential confounders. Results Bicycle commuters had significantly lower risk of being stressed than non-bicycle commuters (Relative Risk; RR (95% CI)=0.73 (0.60 to 0.89), p=0.001). Bicycle commuters who bicycled 4 days per week (RR (95% CI)=0.42 (0.24 to 0.73), p=0.002) and those who bicycled 5 or more days per week (RR (95% CI)=0.57 (0.42 to 0.77), p<0.001) had lower risk of being stressed than those who bicycled less than 4 days. This relationship remained statistically significant after adjusting for individual and environmental confounders and when using different cut-offs of perceived stress. Conclusions Stress reduction may be an important consequence of routine bicycle use and should be considered by decision makers as another potential benefit of its promotion.