Columnaris disease is a major problem for the aquaculture industry worldwide and results in substantial mortality and economic loss. The pathogenesis of the disease is poorly understood, and the existing control measures are ineffective. Flavobacterium columnare causes columnaris disease in wild, farmed, and ornamental freshwater fishes. Challenge experiments revealed that the type IX protein secretion system (T9SS) is required for virulence. When a core component of this system was deleted, the mutant was deficient in secretion and in gliding motility, and was avirulent towards zebrafish, rainbow trout, and channel catfish. The T9SS secretes many proteins. Analysis of cell-free spent culture fluid of wild type and T9SS mutant cells identified more than 50 proteins that are secreted by the T9SS. Among these were predicted adhesins, cytolysins, proteases, chondroitin sulfate lyases, and proteins involved in gliding motility and in iron uptake. We deleted genes encoding proteins secreted by the T9SS, including the motility adhesin SprB, chondroitin sulfate lyases, and proteases, to identify those critical for virulence. Cells lacking SprB, or cells lacking chondroitin sulfate lyases, were partially defective in virulence. In immersion challenges, the sprB mutant exhibited reduced mortality of zebrafish, and the chondroitin sulfate lyase mutant exhibited reduced mortality and reduced fin damage compared to fish exposed to a similar dose of the wild type strain. Mutants lacking individual proteases were as virulent as wild type cells, but a mutant lacking ten protease-encoding genes exhibited decreased virulence in zebrafish immersion challenges. The results suggest that multiple secreted proteins may be important for virulence. SprB facilitates attachment and movement of F. columnare along surfaces. It may be important for colonization of fish tissues and formation of the characteristic haystacks on infected gills. The sprB mutant formed non-spreading, non-rhizoid colonies on agar, and others have demonstrated a correlation between rhizoid colony morphology and virulence. Chondroitin sulfate lyases degrade chondroitin sulfate found in cartilage and connective tissues. Decreased levels of this enzyme resulted in decreased digestion of fins, a common site of F. columnare infections. Secreted proteases may digest proteins of the extracellular matrix and may contribute to the necrotic skin, gill, and muscle lesions associated with columnaris disease. F. columnare produces many proteases, and redundancy may explain the virulence of many of our single and multiple protease deletion mutants. Vaccination trials are underway to evaluate the immunizing potency of these mutants in zebrafish under controlled laboratory settings. Zebrafish survivors (previously exposed to ΔsprB mutant) exhibited modest resistance to infection by wild type cells. Future experiments aim to develop an effective vaccine and to design sustainable strategies to control columnaris disease in aquaculture.