Abstract Proteomics and fermentation technology have begun to integrate to investigate fermentation organisms in bioprocess development. This is the first shotgun proteomics study employed to monitor the proteomes of Scheffersomyces stipitis during xylose fermentation under oxygen limitation. We identified 958 nonredundant proteins and observed highly similar proteomes from exponential to early stationary phases. In analyzing the temporal proteome, we identified unique expression patterns in biological processes and metabolic pathways, including alternative respiration salicylhydroxamic acid (SHAM) pathway, activation of glyoxylate cycle, expression of galactose enzymes, and secondary zinc-containing alcohol dehydrogenase and O-glycosyl hydrolases. We identified the expression of a putative, high-affinity xylose sugar transporter Xut1p, but low-affinity xylose transporters were absent. Throughout cell growth, housekeeping processes included oxidative phosphorylation, glycolysis, nonoxidative branch of the pentose phosphate pathway, gluconeogenesis, biosynthesis of amino acids and aminoacyl total RNA (tRNA), protein synthesis and proteolysis, fatty acid metabolism, and cell division. This study emphasized qualitative analysis and demonstrated that shotgun proteomics is capable of monitoring S. stipitis fermentation and identifying physiological states, such as nutrient deficiency.