Proteases are the group of enzymes that carry out proteolysis in all forms of life and play an essential role in cell survival. By acting on specific functional proteins, proteases affect the transcriptional and post-translational pathways in a cell. Lon, FtsH, HslVU and the Clp family are among the ATP-dependent proteases responsible for intracellular proteolysis in bacteria. In bacteria, Lon protease acts as a global regulator, governs an array of important functions such as DNA replication and repair, virulence factors, stress response and biofilm formation, among others. Moreover, Lon is involved in the regulation of bacterial metabolism and toxin–antitoxin systems. Hence, understanding the contribution and mechanisms of Lon as a global regulator in bacterial pathogenesis is crucial. In this review, we discuss the structure and substrate specificity of the bacterial Lon protease, as well as its ability to regulate bacterial pathogenesis.