CONTEXT: Exercise is recommended for postpartum health, but its impacts on breastmilk composition and offspring are understudied. OBJECTIVE: To test whether the breastmilk metabolome is altered with (i) acute exercise and/or (ii) habitual physical activity, and (iii) whether exercise-altered metabolites are associated with infant adiposity. DESIGN: Milk metabolites were assessed before and after acute exercise and in association with habitual activity score in two independent cohorts. SETTING: Two academic medical centers. PARTICIPANTS: The acute exercise cohort had 15 mother-infant dyads. The habitual activity nested case-control analysis had 84 physically active 'cases' and 35 inactive 'controls', and was done in a subset of the MILk/4M Study (N=348). INTERVENTIONS/EXPOSURES: The acute exercise exposure was a 30-minute moderate-intensity treadmill session. The habitual activity exposure was based on Physical Activity Recall questionnaire scores. MAIN OUTCOME MEASURES: Milk metabolite relative abundance at 1-month postpartum by LC/GC-MS, infant anthropometric and body composition measures at 1, 3, and 6 months. RESULTS: An acute exercise bout altered milk concentrations in 28 of 511 detectable metabolites (FDR<0.05). In the habitual activity analysis, 4 of 454 detectable metabolites differed between active cases vs. inactive controls (FDR<0.05). Ten metabolites were altered (p<0.05) by both exercise exposures. Of these, 4 were positively associated with fat mass index at 1 month, and 2 were associated with greater increase in BMI z-score between 1-3 months. CONCLUSIONS: Maternal exercise was associated with differences in the breastmilk metabolome. Metabolites that were associated with both acute exercise and habitual activity correlated with infant adiposity measures.