Understanding the cellular response mechanisms of Candida tropicalis is crucial to biofuel production from corn stover, and targeted genetic modification of a C. tropicalis fermentation strain can improve the biofuel yield. In this report, metabolomic analysis of a hydrolysate obtained from dilute acid hydrolysis of corn stover identified 1,469 substances, including sugars, aldehydes, acids and phenols. In the presence of the corn stover hydrolysate, cell growth was inhibited. Moreover, subcellular observations revealed that C. tropicalis SHC-03 accumulated reactive oxygen species and maintained endoplasmic reticulum homeostasis when incubated with this hydrolysate. For detoxification of byproducts in the hydrolysate that inhibit cell growth and survival, genes associated with reduced glutathione, ergosterol, and ubiquinone-n biosynthesis and misfolded protein and fatty acid degradation were upregulated upon incubation with the hydrolysate. These results will help guide genetic modifications that increase the intracellular synthesis of NADH/NADPH and acetyl-CoA for ergosterol and fatty acid accumulation to improve tolerance to hydrolytically toxic byproducts and accelerate industrial production of bioethanol and other bioproducts.