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Limited data on the relationship between physical activity and lipoprotein particle profiles exist. Our objective was to investigate associations between objectively measured physical activity and lipoprotein particle size and number, and specifically whether substituting daily sedentary behavior with light activity or moderate-to-vigorous physical activity (MVPA) is associated with beneficial alterations to the lipoprotein profile among adults and those at increased cardiometabolic risk (obese and insulin-resistant subjects).Sedentary behavior and physical activity intensity and duration were measured for 7 consecutive days using the GENEActiv accelerometer in a cross-sectional adult cohort (n = 396; mean age, 59.6 ± 5.5 yr). Lipoprotein particle size and subclass concentrations were determined using nuclear magnetic resonance spectroscopy. Isotemporal substitution regression modeling quantified the associations between replacing 30 min·d−1 of sedentary behavior with equal amounts of light activity and MVPA on lipoprotein profiles.Daily duration of MVPA was inversely associated with large VLDL particles and lipoprotein insulin resistance scores (P < 0.05, after adjustment for sedentary time and other confounding factors). Reallocating 30 min of sedentary time with MVPA, but not light activity, was associated with less large VLDL particles resulting in more favorable average VLDL particle size and improved lipoprotein insulin resistance score (P < 0.05). Analysis of high-cardiometabolic-risk groups revealed similar beneficial alterations to VLDL profiles (P < 0.05) with substitution of sedentary time for MVPA among the insulin-resistant (homeostasis model assessment for insulin resistance ≥75th percentile) but not the obese (body mass index ≥30 kg·m−2) individuals.Daily MVPA duration and theoretical replacement of sedentary time with MVPA, but not light activity, were associated with less atherogenic VLDL profiles, particularly among the insulin-resistant individuals. These findings, which require further investigation, highlight the need to develop physical activity interventions aimed at improving atherogenic dyslipidemia and lowering cardiometabolic risk.