High-density lipoproteins (HDL) across all species, are largely known for their role in cholesterol transport. Human HDL contain over 95 different proteins, and only one-third are dedicated to lipid transport. The HDL proteome consists largely of proteins with anti-oxidant, complement regulatory, proteolytic, or antithrombotic activity. Several physiologic and pathologic states are associated with changes in the HDL proteome. The transition period in a dairy cow, a high-risk time for development of production-related diseases, is marked by profound changes in lipid metabolism. In this study, quantitative proteomic analysis was performed on HDL samples from 28 clinically healthy, multiparous Holstein cows at 2 weeks prepartum (wk −2), week of calving (wk 0) and 2 wk postpartum (wk +2). HDL were isolated by density gradient ultracentrifugation. Samples were equivalently loaded (75 µg protein) with N15 labeled human ApoA1 as an additional loading control and analyzed using ESI-MS/MS QStar XL mass spectrometer (Applied Biosystem). Generated peaks were scanned against the UniProtKB/Swiss-Prot Protein Knowledgebase (release 57.0, 03/2009) using the X!Tandem search engine. Relative protein counts were analyzed by repeated measures ANOVA. 81 unique sequences were identified (95% probability match). The majority of proteins were similar to those identified on human HDL with the salient exception of glycosylation dependent cell adhesion molecule 1, which has not been found on human HDL. Relative protein count of apolipoprotein CII (ApoCII) was significantly different across all time points studied (0.9 wk −2, 2.8 wk 0, 4.7 wk +2, P < 0.05, n = 4 per time point). ApoCII is postulated to have lipopolysaccharide-neutralizing effects. Relative amount of serum amyloid A was also divergent (0.4 wk −2, 1.8 wk 0, 1.0 wk +2, P < 0.05, n = 4 per time point), with smaller changes noted in α 1 antiprotease, paraoxonase, and 3 different glycoproteins. Proportion of HDL was not different across time points (75.7% ± 5.4 wk −2, 83.7% ± 6.5 wk 0, 85.3% ± 3.8% wk+2, P > 0.06). In addition to the well-recognized decline in HDL that occurs during transition, our data indicate the composition of HDL varies across this time as well.