Gram-negative bacteria harness multiple protein secretion systems and secrete a large proportion of the proteome. Proteins can be exported to periplasmic space, integrated into membrane, transported into extracellular milieu, or translocated into cytoplasm of contacting cells. It is important for accurate, genome-wide annotation of the secreted proteins and their secretion pathways. In this review, we systematically classified the secreted proteins according to the types of secretion systems in Gram-negative bacteria, summarized the known features of these proteins, and reviewed the algorithms and tools for their prediction.