In spite of the difficulties in delivering PUFA to ruminants, studies have generally indicated that the PUFA of the omega-6 (linoleic acid) and omega-3 [α-linolenic acid; eicosapentaenoic (EPA), C20:5 omega-3; docosahexaenoic (DHA), C22:6 omega-3] families are the most beneficial to improving reproduction in cows. The objectives were to determine if a diet enriched in α-linolenic acid (omega-3) or linoleic acid (omega-6) would influence milk production and composition, metabolic status, and reproductive performance in lactating dairy cows. High-yielding multiparous Holstein dairy cows (n = 120) with no overt clinical illnesses were blocked according to calving date and parity. Cows were assigned randomly to be fed 1) soybean whole roast (Soy, omega-6, n = 40) or 2) linseed (Lin, omega-3, n = 40) or 3) palm oil as a source of SFA (PO, n = 40) from calving until first heat after 40 d postpartum (dpp), and then half of the cows in each treatment group were switched to receive either Lin or SFA (PO) from first heat after d 40 to 120 dpp. Blood was collected from a subsample of cows. Blood was collected at 14 d intervals for 12 wk, starting on the day of calving. Results showed milk yield and DMI were not affected. Milk compositions were similar (P > 0.08) among diets, except concentration and yield of milk fat percentage, which was less in cows fed Lin (P < 0.05). Uterine involution in cows fed Soy occurred earlier (P < 0.05). Diets affected day to first estrus and day to first insemination in cows (P < 0.05). There were no differences among treatments for percent heat detection, percent pregnancy per first insemination, and percent conception per AI at estrus. Also, there is a trend of pregnancy by 120 d, which is 66.7% for the Lin group vs. 50.91% for the PO group (P < 0.08). Of the 4 pregnancy losses, 2 occurred in PO-PO group and 2 occurred in Soy-PO group, and none occurred in the other 4 treatments. In conclusion, our study showed feeding omega-6 fatty acids during 40 dpp could be a good treatment for early postpartum periods, and a shift to omega-3 fatty acids until 40 d after AI can be considered as a strategy for improving fertility in lactating dairy cows.