This study aimed to evaluate for the first time the temporal dynamic changes in early postmortem proteome of normal and high ultimate pH (pHu) beef samples from the same cattle using a shotgun proteomics approach. Ten selected carcasses classified as normal (pHu < 5.8; n = 5) or high (pHu ≥ 6.2; n = 5) pHu beef from pasture-finished Nellore (Bos taurus indicus) bulls were sampled from Longissimus thoracis muscle at 30 min, 9 h and 44 h postmortem for proteome comparison. The temporal proteomics profiling quantified 863 proteins, from which 251 were differentially abundant (DAPs) between high and normal pHu at 30 min (n = 33), 9 h (n = 181) and 44 h (n = 37). Among the myriad interconnected pathways regulating pH decline during postmortem metabolism, this study revealed the pivotal role of energy metabolism, cellular response to stress, oxidoreductase activity and muscle system process pathways throughout the early postmortem. Twenty-three proteins overlap among postmortem times and may be suggested as candidate biomarkers to the dark-cutting condition development. The study further evidenced for the first time the central role of ribosomal proteins and histones in the first minutes after animal bleeding. Moreover, this study revealed the disparity in the mechanisms underpinning the development of dark-cutting beef condition among postmortem times, emphasizing multiple dynamic changes in the muscle proteome. Therefore, this study revealed important insights regarding the temporal dynamic changes that occur in early postmortem of high and normal muscle pHu beef, proposing specific pathways to determine the biological mechanisms behind dark-cutting determination. •In-depth proteomics exploration of dark-cutting beef condition using three muscle sampling times taken early postmortem.•251 proteins were differentially abundant creating the most comprehensive interconnected pathways of high-pHu beef.•Energy metabolism, cellular stress response and oxidoreductase activity play pivotal roles.•Ribosomal proteins and histones play pivotal role in dark-cutting beef development.•Pathways are proposed to determine the biological mechanisms behind dark-cutting beef of Nellore cattle.