Dietary fatty acids (FA) are directly transferred to milk through chylomicrons and indirectly through tissue recycling. The objective of this study was to characterize the direct and indirect transfer rates of the cis-9,trans-11 (c9t11) and trans-10,cis-12 (t10c12) CLA through plasma to milk following a single abomasally infused bolus. Five ruminally cannulated multiparous mid-lactation cows (148 ± 86 DIM; Milk 44.1 ± 11.2 kg/d) received a single abomasal bolus infusion of an enriched CLA mixture providing 15 g of each CLA isomer (c9t11, t10c12) over a 30 min period. Total transfer of CLA was analyzed in a model that included cow as a random effect and CLA isomer as a fixed effect (JMP Pro). Time course data were analyzed as repeated measures in SAS and least squares means were fit to a double exponential decay function by nonlinear curve fitting (JMP Pro) to characterize direct (fast pool) and indirect (slow pool) transfer of CLA isomers to milk. Plasma CLA concentration peaked at 2 h, reaching 0.32 and 0.31% of plasma FA for c9t11 and t10c12, respectively, and returned to baseline at 72 h. Milk t10c12 concentration peaked at 14 h (0.5% of FA) and returned to baseline at 86 h post infusion. Milk c9t11 concentration initially peaked at 14 h (0.98% of FA), returned to baseline at 86 h post infusion, and then had a second peak between 146 to 158 h (0.56% of FA) post infusion. Total transfer of CLA to milk differed between isomers and was 79.3 and 40.8% of the bolus for c9t11 and t10c12, respectively (P < 0.001). Time course of CLA isomers transferred to milk fit a biexponential model (R² = 0.99). The area (% of total) under the first exponential representing direct transfer was 17 and 73% and the second exponential representing indirect transfer was 83 and 27% of the total CLA isomers transferred for c9t11 and t10c12, respectively. In conclusion, although plasma kinetics of c9t11 and t10c12 were similar, transfer of CLA isomers to milk differed greatly in their transfer efficiency and major pool of transfer.