Melatonin, a hormone secreted in cow milk, has beneficial effects on human health. This study aimed to evaluate the effects of dietary vitamin supplementation and nocturnal milking on the concentration of melatonin in cow milk. Twenty-one confined cows in the final period of lactation were used for the study and the supplemented vitamins were: rumen-protected B vitamins, A, D3, E, and niacin (B3) not protected from rumen degradation. The experimental period had a duration of 10 days. Milk samples were collected during the first two days, without supplementation of vitamins (WITHOUT), and during the final two days, eight days after the beginning of supplementation (WITH). Samples of milk produced without and with supplementation, milked at 05:00 h (NIGHT) or from the total milk produced in the two daily milkings (TOTAL), were analyzed. The concentration of melatonin was determined by enzyme immunoassay using the ELISA kit, and the results were analyzed by ANOVA and Tukey´s test with 5% significance. Supplementation of the diet with vitamins (WITH) did not affect melatonin concentration in TOTAL milk, but it resulted in a concentration 40.55% lower in the NIGHT milk than in the milk from cows without (WITHOUT) supplementation (6.57 vs. 11.06 pg mL-1). Milk milked at night showed melatonin concentrations 1.43 to 2.38 times higher than daily TOTAL milk. The total milk of the herd showed a concentration of 2.13 pg mL-1. The differentiated collection of milk produced during the night period makes it possible to obtain milk with high levels of melatonin.