Omega-3 (n-3) fatty acids (FA) are essential nutrients and their metabolism has not been characterized across lactation in the cow. The objective of this study was to determine the effect of lactation stage on transfer of n-3 FA to plasma and milk following a bolus infusion. Four ruminally cannulated multiparous Holstein cows received a single abomasal bolus infusion of an enriched n-3 FA mixture (flaxseed, fish and algae oil) in early (10–20 DIM), peak (50–60 DIM), and late (>200 DIM) lactation. Treatments provided 50 g of α-linolenic acid (ALA), 7 g of eicosapentaenoic acid (EPA), and 20 g of docosahexaenoic acid (DHA), as free FA over a 30 min period. Total plasma and milk FA were analyzed over 10 d starting a day before treatments. Total transfer to milk was analyzed in a model that included random effect of cow nested in lactation stage and fixed effect of lactation stage (JMP Pro). Time course data relative to bolus was analyzed as repeated measures in SAS with cow nested in lactation stage as random effect and lactation stage, time, and their interaction as fixed effects. Lactation stage and its interaction with time was significant for total plasma ALA, EPA and DHA, and for milk ALA and DHA concentration (all P < 0.001). Plasma ALA peaked at 12, 6, and 4 h post bolus at 5.9, 5.3, and 4.8% of total FA in early, peak, and late lactation, respectively. Plasma EPA peaked at 12 h (all stages) at 2.2, 1.3, and 1% of total FA, while plasma DHA peaked at 6, 6, and 4 h post bolus at 1.1, 0.6, and 0.5% of total FA in early, peak, and late lactation, respectively. Milk ALA and DHA (%) peaked at 14 h in all stages, while milk EPA peaked 26 h post bolus in early lactation and 14 h in peak and late lactation. Transfer of ALA to milk reached 59% and did not differ by stage; while transfer of EPA was 90% higher in peak and late lactation compared with early lactation (34% vs. 18%, P < 0.05). Transfer of DHA reached 32% in peak lactation, which was 42% and 29% higher than transfer at early and late lactation, respectively (P < 0.05). In conclusion, enrichment of plasma and milk, and transfer efficiencies of n-3 FA differs between stages of lactation, which may indicate specific n-3 FA requirements or metabolism for each stage of lactation.