Serine proteases exist in eukaryotic and prokaryotic organisms and have emerged during evolution as the most abundant and functionally diverse group. In Gram-negative bacteria, there is a growing family of high molecular weight serine proteases secreted to the external milieu by a fascinating and widely employed bacterial secretion mechanism, known as the autotransporter pathway. They were initially found in Neisseria, Shigella, and pathogenic Escherichiacoli, but have now also been identified in Citrobacterrodentium, Salmonella, and Edwardsiella species. Here, we focus on proteins belonging to the serine protease autotransporter of Enterobacteriaceae (SPATEs) family. Recent findings regarding the predilection of serine proteases to host intracellular or extracellular protein-substrates involved in numerous biological functions, such as those implicated in cytoskeleton stability, autophagy or innate and adaptive immunity, have helped provide a better understanding of SPATEs’ contributions in pathogenesis. Here, we discuss their classification, substrate specificity, and potential roles in pathogenesis.