Mammalian cells are the preferred expression system for obtaining recombinant proteins (r-proteins) due to their ability to generate human-like glycosylation patterns. However, their slow growth and lower productivity compared to prokaryotic host cells, coupled with the rising demand for complex therapeutic proteins in the biopharmaceutical market, have driven the search for alternatives to boost productivity. In this context, mild hypothermia (MH) has emerged as a valuable tool for enhancing both the viability and productivity of r-proteins in mammalian as well as some insect-derived cell lines. Notably, the impact of MH varies depending on the r-protein and cell line, and is influenced by factors such as promoter type, temperature reduction methods, supplementation, pH, and operational conditions. MH can affect substrate synthesis, toxic metabolite production, and post-translational modifications of r-proteins, particularly glycosylation. At the molecular level, MH influences processes such as cell cycle arrest, apoptosis delay, mRNA stability, protein synthesis, cytoskeletal reorganization, and the induction of endogenous transcription factors, all of which can contribute to increased productivity and viability in cell cultures. This review addresses key considerations regarding the application of MH in mammalian and insect cell cultures and provides a comprehensive overview of the molecular mechanisms underlying its effects. It also identifies potential targets for cell engineering that could further enhance r-protein production.