Our understanding of rumen microbial diversity and the biochemical pathways performed by specific microbe populations is expanding; however, there is a distinct lack in the use of proteomic techniques to progress our knowledge of shifts in magnitude and pattern of these protein-mediated pathways. It was hypothesized that within a 24-h period, there would be fluctuations of rumen microbial protein abundances due to feed intake-mediated nutrient availability. This study investigated the fluctuations of bovine rumen metaproteome over a 24-h period utilizing 3 mid to late-lactation Holsteins (DIM = 219.3 ± 77.7) that were fed the same TMR ad libitum. Rumen fluid was collected on 3 d within a 5-d period at 3 time points throughout the day relative to their first offering of TMR (0h, 4h, and 6h). Samples were pooled within time point within cow across day, analyzed using LC-MS/MS techniques, and analyzed for variations across hour of sampling using PROC MIXED of SAS. A total of 256 proteins were characterized across 12 microbial species, with 21 proteins identified from a variety of 7 species affected by time of collection. From 4 of these species, 8 50S ribosomal protein subunits were affected by hour of sampling, with 7 out of 8 of these subunits increased in abundance over the 3 time points. Ruminococcus flavefaciens 007c had lower abundance of a nitrogen fixing protein (P = 0.04) as hour of sampling increased, while 3 other species expressed a decrease in abundance of gluconeogenesis-involved proteins GAPDH (P = 0.04) and PPDK (P = 0.003 and P = 0.026) as hour of sampling increased. Protein abundances of cysteine synthase (P = 0.033; F. succinogenes) and O-acetylhomoserine sulfhydrolase (P = 0.037; T. saccharophilum), 2 proteins involved in amino acid synthesis, were negatively correlated with hour of sampling. Results suggest that as nutrients become more readily available, microbes shift from conversion-focused biosynthetic routes to more encompassing DNA-driven pathways.