This study investigates the impact of mild hypothermic conditions (31°C) on the whole cell proteome and ubiquitinated proteome of Chinese hamster ovary (CHO) cells, with a specific focus on endoplasmic reticulum (ER) proteins and ER stress. Using high-resolution mass spectrometry, we conducted a comprehensive proteomic and ubiquitinated proteomic analysis to quantify changes in protein abundance and ubiquitinated peptides under mild hypothermia.