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Objective: Higher N-terminal pro-brain natriuretic peptide (NT-proBNP) levels are a strong risk factor for cardiovascular disease. The current study aimed to clarify the cross-sectional association of physical activity (PA) with NT-proBNP and to identify the interaction of PA with alcohol consumption or cigarette smoking in middle-aged individuals., Methods: The study included 4613 individuals (1824 men and 2789 women) (November 2005-November 2006). Total PA, steps, light-intensity PA (LPA), and moderate-to-vigorous-intensity PA (MVPA) were assessed using accelerometer. Serum NT-proBNP levels were measured. Cross-sectional associations of total PA and steps with NT-proBNP were analyzed using multiple regression with adjustment for potential confounders. The isotemporal substitution model was used to assess activity intensity-specific association. The interaction between PA and alcohol consumption or smoking was also examined., Results: Total PA was independently and inversely associated with NT-proBNP in the entire sample (P = 0.04). The inverse association of substituting LPA with MVPA for NT-proBNP was clearer in men than in women (P interaction  = 0.04). Inverse associations of total PA or steps with NT-proBNP were clearer in heavy drinkers than in moderate drinkers and non-drinkers in the entire sample (P interaction  < 0.05). In men, the inverse association of substituting LPA with MVPA for NT-proBNP was also clearer in heavy drinkers (P interaction  = 0.02). No interactions of PA with smoking were detected., Conclusions: Higher total PA was associated with better NT-proBNP in middle-aged individuals. Additionally, the effect of substituting LPA with MVPA on NT-proBNP was greater in men than in women. Furthermore, the association between PA and NT-proBNP may be modified by alcohol consumption., Competing Interests: Declaration of competing interest The authors declare no conflicts of interest in association with the present study., (Copyright © 2024 Elsevier Inc. All rights reserved.)