Dairy cows have a daily rhythm of milk synthesis that appears to be driven by the molecular clock of the mammary gland and is modified by the time of feed availability. Protein metabolism is intimately linked to circadian rhythms in other species, but the effect of amino acids on the mammary clock in unknown. The objective was to determine the effects of the timing of protein absorption on the daily rhythms of milk synthesis in dairy cows. Nine cannulated lactating Holstein cows (128 ± 46 d postpartum; mean ± SD) were randomly assigned to 1 of 3 treatment sequences in a 3 × 3 Latin square. Treatments were absomasal infusions of 500 g/d of sodium caseinate either 24 h/d (CON) or for 8 h/d from 0900 to 1700 h (DAY) or from 2100 to 0500 (NGT). Treatment periods were 15 d with a 6 d washout. During the final 8 d of each period, cows were milked every 6 h. A 24-h rhythm was fit to time-course production data using cosine analysis and the amplitude and acrophase (time at peak) were determined. Daily milk and protein yield were decreased by NGT compared with CON, while fat yield was increased by DAY (P < 0.05). Daily fat and protein concentration were not affected by treatment. Milk yield failed to fit a 24 h rhythm in CON or DAY, but a rhythm was induced by NGT (P = 0.03). Neither fat yield nor protein yield fit a rhythm in either treatment. Fat concentration fit a daily rhythm in all treatments (P < 0.05), with the amplitude decreased 57% by DAY and 26% by NGT (P < 0.05). The rhythm of milk fat concentration was phase advanced ~2 h by DAY and phase delayed ~1 h by NGT (P < 0.05). Protein concentration fit a daily rhythm in CON and DAY but not NGT. The phase of protein concentration was delayed ~1.25 h by DAY and the amplitude was increased 2-fold relative to CON (P < 0.05). The time of protein infusion influenced daily rhythms of milk and milk protein synthesis. Night infusion abolished rhythms of protein concentration and induced rhythms of milk yield while day infusion increased the amplitude of protein concentration. The results of this study suggest a role of amino acids in entraining the molecular clock of the mammary gland.