This paper provides causal inference on how transport intervention affects moderate-to-vigorous physical activity (MVPA) and walking among older adults using a natural experiment of a new metro line in Hong Kong. A longitudinal survey of 449 cohort participants was collected before and after the metro operation. Treatment groups live within a 400m walking buffer of the new metro stations, while control groups are located around comparable stations on existing metro lines. These metro lines were planned at the same time using similar principles, but the intervention line was built later due to different financial models. Our difference-in-difference (DID) models found that the new metro line significantly decreased older adults' weekly MVPA (−129.33 min, p < 0.05) in treatment groups, while the effect on change in walking time did not significantly differ between the treatment and control groups. We also found heterogeneous treatment effects among gender and age subgroups. Furthermore, our time effect tests suggested that older adults’ physical activity and walking levels may stabilise, based on participants living around a metro station operated four years ago with another comparable station operated three decades ago. This practice-based evidence suggests that new metro developments might not promote physical activity and walking levels among older adults in the high-density city of Hong Kong.