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 (Insig1 and Srebf2) 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, Zmat3 and Btg2, 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.