Circadian rhythms are generated within tissues through a molecular clock made of a set of transcription factors that oscillate in a 24-h manner. These rhythms can be entrained both by photoperiod and feeding time. Dairy cows display daily rhythms of milk synthesis that are altered by feeding time, but the role of molecular clocks in these rhythms is poorly understood. To determine the impact of feeding on the mammary molecular clock, 11 mid-lactation (168 ± 50 DIM) multiparous Holstein cows were used in a crossover design (5 or 6 cows per treatment) with two 25-d periods. Treatments included (1) ad libitum feed available for the entire day (AL), or (2) night-restricted feeding, where feed availability was limited to 16 h/d from 2000 h to 1200 h (NR). All cows were housed in the same 19:5 light:dark cycle. Milk samples were collected at 0700 h and 1900 h on d 11 and d 17 of each period and analyzed for fat and protein concentration. Mammary tissue was collected from all cows via needle biopsy to represent 4 times across the day (0400, 1000, 1600, and 2200 h). The expression of clock genes Bmal1, Clock, Cry1, Per1, Per2, and Rev-erbα was determined at each time point using Real-Time RT-PCR. Cosinor rhythmometry was performed using SAS Proc Mixed to determine if expression of clock genes fit a 24 h rhythm and if the amplitude and acrophase (time at peak) differed between treatments. The NR treatment reduced dry matter intake (1.9 kg), yield of milk (3.0 kg), fat (160 g) and protein (113 g), and milk protein concentration (0.06%; all P < 0.05), while milk fat concentration was not affected (P = 0.91). Daily rhythms of Bmal1, Clock, and Per1 were not observed in either treatment. Cry1 fit a 24 h rhythm in both treatments (P < 0.05), with an increased amplitude and an acrophase occurring 8.5 h later in NR compared with AL. Rev-erbα fit a daily rhythm in the NR treatment (P = 0.03) and tended to fit a rhythm in the AL treatment (P = 0.05). The acrophase of Rev-erbα was shifted 6.5 h later in NR compared with AL, but the amplitude did not differ by treatment. Per2 did not express a daily rhythm in the AL group, but a rhythm was induced in NR cows (P = 0.02). These results indicate that key components of the mammary molecular clock are influenced by feeding time.