Ubiquitin and ubiquitin-like modifiers constitute one of the most versatile systems for the control of protein function by post-translational modifications. While the ubiquitin pathway has been very well characterized, our knowledge of SUMO and its mechanisms of action is still limited. Recent technological developments have contributed to improve the basic knowledge of the biochemistry of the SUMO pathway, as well as to gain insight into the biological roles of protein SUMOylation. Here we dissect the biochemistry behind protein SUMOylation, and we describe the specific characteristics of SUMO that set it apart from the ubiquitin pathway. We cover the machinery involved in SUMO maturation, its conjugation (writers), and removal (erasers), as well as the interplay SUMO-ubiquitin. Then, we focus on the role of SUMO-interacting motifs (readers) in the recognition of protein SUMOylation, the formation of SUMO hubs, and the induction of phase separation and condensates. Last, we outline the functions of SUMO in physiology and in pathological conditions such as cancer. Our manuscript summarizes the essential knowledge about the biochemistry and regulation of the SUMO pathway.