Obesity is associated with chronic inflammation and is a risk factor for insufficient milk production. Inflammation-mediated suppression of LPL could inhibit mammary uptake of long-chain fatty acids (LCFAs; >16 carbons). In an ancillary case-control analysis, we investigated whether women with low milk production despite regular breast emptying have elevated inflammation and disrupted transfer of LCFAs from plasma into milk. Data and specimens from a low milk supply study and an exclusively breastfeeding control group were analyzed, with milk production measured by 24-h test-weighing at 2-10 wk postpartum. Low milk supply groups were defined as very low (VL; <300mL/d; n=23) or moderate (MOD; ≥300mL/d; n=20) milk production, and compared with controls (≥699mL/d; n=18). Serum and milk fatty acids (weight% of total) were measured by GC, serum and milk TNF-α by ELISA, and serum high-sensitivity C-reactive protein (hsCRP) by clinical analyzer. Group differences were assessed by linear regression models, chi-square exact tests, and Kruskal-Wallis nonparametric tests. VL cases, as compared with MOD cases and controls, had higher prevalence of elevated serum hsCRP (>5mg/L; 57%, 15%, and 22%, respectively; P=0.004), detectable milk TNF-α (67%, 32%, and 33%, respectively; P=0.04), and obesity (78%, 40%, and 22%, respectively; P=0.003). VL cases had lower mean±SD LCFAs in milk (60%±3%) than MOD cases (65%±4%) and controls (66%±5%) (P<0.001). Milk and serum LCFAs were strongly correlated in controls (r=0.82, P<0.001), but not in the MOD (r=0.25, P=0.30) or VL (r=0.20, P=0.41) groups (Pint<0.001). Mothers with very low milk production have significantly higher obesity and inflammatory biomarkers, lower LCFAs in milk, and disrupted association between plasma and milk LCFAs. These data support the hypothesis that inflammation disrupts normal mammary gland fatty acid uptake. Further research should address impacts of inflammation and obesity on mammary fatty acid uptake for milk production.