BACKGROUND: Health and wellbeing are partly shaped by the neighbourhood environment. In 2011, an eight kilometre (five mile) extension to the M74 motorway was opened in Glasgow, Scotland, constructed through a predominantly urban, deprived area. We evaluated the effects of the new motorway on wellbeing in local residents. METHODS: This natural experimental study involved a longitudinal cohort (n=365) and two cross-sectional samples (baseline n=980; follow-up n=978) recruited in 2005 and 2013. Adults from one of three study areas — surrounding the new motorway, another existing motorway, or no motorway — completed a postal survey. Within areas, individual measures of motorway proximity were calculated. Wellbeing was assessed with the mental (MCS-8) and physical (PCS-8) components of the SF-8 scale at both time points, and the short Warwick-Edinburgh Mental Well-being Scale (SWEMWBS) at follow-up only. RESULTS: In multivariable linear regression analyses, cohort participants living nearer to the new M74 motorway experienced significantly reduced mental wellbeing over time (MCS-8: -3.6, 95% CI -6.6 to -0.7) compared to those living further away. In cross-sectional and repeat cross-sectional analyses, an interaction was found whereby participants with a chronic condition living nearer to the established M8 motorway experienced reduced (MCS-8: -3.7, 95% CI -8.3 to 0.9) or poorer (SWEMWBS: -1.1, 95% CI -2.0 to -0.3) mental wellbeing compared to those living further away. CONCLUSIONS: We found some evidence that living near to a new motorway worsened local residents’ wellbeing. In an area with an existing motorway, negative impacts appeared to be concentrated in those with chronic conditions, which may exacerbate existing health inequalities and contribute to poorer health outcomes. Health impacts of this type of urban regeneration intervention should be more fully taken into account in future policy and planning.