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ABSTRACT Chinese hamster ovary (CHO) cells are widely used in recombinant biopharmaceutical production; yet, yields remain low, leading to high market prices. Improving product yield and quality has heavily relied on empirical characterization with limited insight into internal molecular dynamics. RNA‐seq offers a powerful alternative to understand intracellular responses to process changes through gene expression measurement. In this study, three RNA‐seq datasets across three CHO cell lines and four industrially relevant treatments were integrated to characterize the global transcriptome changes, construct a weighted gene co‐expression network, assess the impact on recombinant anti‐interleukin 8 (anti‐IL8) immunoglobulin heavy and light chain transcript abundance, and expression of glycosylation genes. Treatments included adaptation to serum‐free medium, low temperature, low pH, and low glucose concentration in the medium. The findings suggest upregulation of cholesterol biosynthesis is critical for serum‐free medium adaptation, and the rate‐limiting enzymes in the sterol regulatory element‐binding protein pathway (Insig1andSrebf2) could be targeted to accelerate adaptation. Temperature‐induced cell cycle suppression was likely mediated by p53 activation, consistent with previous reports, with the p53‐targets,Zmat3andBtg2, identified as key hub genes. Conversely, glucose and pH were observed to have negligible impacts on the transcriptome. This study uniquely identifies novel genes mediating temperature‐induced cell cycle arrest, distinct glycosylation‐related gene responses impacting product quality, and new stable housekeeping genes for accurate gene expression normalization in CHO cells.