Proline dehydrogenase (ProDH), also called proline oxidase (POX), is a universal enzyme in living organisms. It catalyzes the oxidation of L-proline to delta1-pyrroline-5-carboxylate leading to the release of electrons, which can be transferred to either electron transfer systems or to molecular oxygen. ProDH is not only essential for proline catabolism but also plays key roles in providing energy, shuttling redox potential between cellular compartments and reactive oxygen species production. Structural analysis of prokaryotic ProDHs already gives some insights into the biochemical activity and biological functions of this enzyme, which can be extended to eukaryotic ProDHs based on sequence similarities. Here we report the most recent investigations on the biochemical and regulation of ProDH at transcriptional, post-transcriptional and translational levels. The biological roles of ProDH in cell homeostasis and adaptation through energetic, developmental, adaptive, physiological and pathological processes in eukaryotes are presented and discussed to create a framework for future research direction.