Excessive maternal fat mass may impair lactogenesis and lead to lower breastmilk volume. We investigated this relationship in rural Indonesian exclusively breastfeeding mother-infant (2-5.3 months) dyads (n = 112) by measuring breastmilk intake by deuterium oxide dose-to-mother technique (DDMT) and maternal fat mass by DDMT, bioelectrical impedance analysis (BIA), and body mass index (BMI). We also compared fat mass assessed by DDMT and BIA. In this population, we found a significant negative relationship between breastmilk intake and maternal fat mass measured by DDMT (β = -5.04 mL, 95% CI: -9.36, -0.72, P = 0.023), and similar but slightly weaker negative trend with BIA and BMI, after adjusting for social-economic status, maternal age, infant age and sex. Maternal fat mass estimates by BIA and DDMT showed good agreement. In light of the trend for overweight and obesity worldwide, further research is needed into the underlying mechanisms of this negative relationship.