Chinese hamster ovary (CHO) is the most commonly used host cell line for therapeutic protein production. Their exposure to highly concentrated feed solution during fed-batch cultivation can cause an unphysiological osmolality increase (>300 mOsm/kg) affecting cell physiology, morphology, and proteome. In a companion article “Hyperosmolality in CHO Culture: Effects on Cellular Behavior and Morphology” we show that hyperosmolalities of up to 545 mOsm/kg force cells to ablate proliferation and gradually increase their volume, almost triplicating it. CHO cells also exhibit a significant hyperosmolality-dependent mitochondrial activity increase. To get a deeper insight into molecular mechanisms involved in these processes, we performed a comparative quantitative label-free proteome study of hyperosmolality-exposed vs. control CHO cells. Our analysis revealed key differentially expressed proteins mediating mitochondrial activation, oxidative stress amelioration, and cell cycle progression. We also discovered a previously unknown strong regulation of proteins altering cell membrane rigidity and permeability. Among others, we detected three members of septins, filamentous proteins forming diffusion barriers in the cell, to be highly upregulated in response to hyperosmolality. Taken together, our observations correlate well with the recent CHO-based fluxome and transcriptome studies and expose new unknown targets involved in response to hyperosmotic pressure in mammalian cells.