New antibiotics with novel modes of action and structures are urgently needed to combat the emergence of multidrug-resistant bacteria.  Bacterial signal peptidase I (SPase I) is an indispensable enzyme responsible for cleaving the signal peptide of preprotein to release the matured proteins.  Increasing evidence suggests that SPase I plays a crucial role in bacterial pathogenesis by regulating the excretion of a variety of virulent factors, maturation of quorum sensing factor and the intrinsic resistance against β-lactams.  Recently, breakthrough has been achieved in the understanding of three-dimensional structure of SPase I as well as the mechanism of enzyme-inhibitors interaction.  Three families of inhibitors are identified, i.e. signal peptide derivatives, β-lactams and arylomycins.  In this article, we summarize the recent advance in the study of structure, activity and structure-activity relationship of SPase I inhibitors.