CLA refers to isomers of octadecadienoic acid with conjugated double bonds. The most abundant form of CLA (rumenic acid (RA): c9,t11-18:2) is found in milk and beef fat. Further, CLA supplements containing RA and t10,c12-18:2 are now available. Consumption of commercially produced CLA has been shown to decrease adipose accretion in growing laboratory and production animals and cause milk fat depression in cows. We tested the hypothesis that CLA supplementation would increase milk CLA concentration and decrease milk fat content in humans. Breastfeeding women (n = 9) participated in this double-blind, placebo-controlled, crossover study divided into three periods: intervention I (5 d), washout (7 d), and intervention II (5 d). Women were randomized to treatment order. During each intervention period, women consumed 1.5 g of CLA supplement or placebo (olive oil) daily; during the washout period, no supplements were consumed. Milk was collected by complete breast expression on the final day of each period; milk output was estimated by 24-h weighing on the penultimate day of each intervention period. Milk RA and t10,c12-18:2 concentrations were greater (P < 0.05) during the CLA treatment period as compared to the placebo period. Milk fat content was significantly lower during the CLA treatment, as compared to the placebo treatment (P < 0.05). Data indicate no effect of treatment on milk output. Therefore, it would be prudent that lactating women not consume commercially available CLA supplements at this time.