We studied the effects of the frequently used glucogenic dietary supplementation in dairy herds and the hormonal changes occurring during the normal estrous cycle on the composition and concentration of milk lipid components. Holstein dairy cows were synchronized with two injections of prostaglandin F2α (estrus = day 0). Animals were held as controls or drenched for 11 days (day −3 to day 8 of the cycle) with 850 ml/day liquid propylene glycol (treatment, n = 13 per group). Blood and milk samples were collected on day 1 and 8 of the cycle. In both groups, plasma progesterone concentration increased ∼10-fold between 1 and 8 days post-estrus. Milk fatty acid composition was associated primarily with estrous-cycle day: polyunsaturated fatty acids increased by 16%, n-6 by 15% and n-3 by 1% from day 1 to 8 post-estrus. Polar lipid composition was also altered by cycle day: phosphatidylethanolamine concentration was 2-fold and 1.5-fold higher on day 1 v. day 8 post-estrus in the control and treatment groups, respectively. Phosphatidylserine concentration in milk was also affected by cycle day by treatment interaction ( P = 0.04). A progesterone level by treatment interaction influenced the triglyceride-to-phospholipid ratio in the milk ( P = 0.02). The results suggest that progesterone plays a role in modulating milk lipid composition and structure. Therefore, strategies designed to alter milk lipid composition should consider the cow's reproductive status.