Grape marc (GPM) is a viticulture by-product that is rich in secondary compounds, including condensed tannins (CT), and is used as a supplement in livestock feeding practices. The aim of this study was to determine whether feeding GPM to lactating dairy cows would alter the milk proteome through changes in nitrogen (N) partitioning. Ten lactating Holstein cows were fed a total mixed ration (TMR) top-dressed with either 1.5 kg dry matter (DM)/cow/day GPM (GPM group; n = 5) or 2.0 kg DM/cow/day of a 50:50 beet pulp: soy hulls mix (control group; n = 5). Characterization of N partitioning and calculation of N partitioning was completed through analysis of plasma urea-N, urine, feces, and milk urea-N. Milk samples were collected for general composition analysis, HPLC quantification of the high abundance milk proteins (including casein isoforms, α-lactalbumin, and β-lactoglobulin) and liquid chromatography tandem mass spectrometry (LC-MS/MS) analysis of the low abundance protein enriched milk fraction. No differences in DMI, N parameters, or calculated N partitioning were observed across treatments. Dietary treatment did not affect milk yield, milk protein or fat content or yield, or the concentrations of high abundance milk proteins quantified by HPLC analysis. Of the 127 milk proteins that were identified by LC-MS/MS analysis, 16 were affected by treatment, including plasma proteins and proteins associated with the blood-milk barrier, suggesting changes in mammary passage. Immunomodulatory proteins, including butyrophilin subfamily 1 member 1A and serum amyloid A protein, were higher in milk from GPM-fed cows. Heightened abundance of bioactive proteins in milk caused by dietary-induced shifts in mammary passage could be a feasible method to enhance the healthfulness of milk for both the milk-fed calf and human consumer. Additionally, the proteome shifts observed in this trial could provide a starting point for the identification of biomarkers suitable for use as indicators of mammary function.