Enterotoxigenic Escherichia coli (ETEC) colonize the human gut, causing severe cholera-like diarrhea. ETEC utilize a diverse array of pili and fimbriae for host colonization, including the Type IVb pilus CFA/III. The CFA/III pilus machinery is encoded on the cof operon, which is similar in gene sequence and synteny to the tcp operon that encodes another Type IVb pilus, the Vibrio cholerae toxin coregulated pilus (TCP). Both pilus operons possess a syntenic gene encoding a protein of unknown function. In V. cholerae, this protein, TcpF, is a critical colonization factor secreted by the TCP apparatus. Here we show that the corresponding ETEC protein, CofJ, is a soluble protein secreted via the CFA/III apparatus. We present a 2.6 Å resolution crystal structure of CofJ, revealing a large β-sandwich protein that bears no sequence or structural homology to TcpF. CofJ has a cluster of exposed hydrophobic side chains at one end and structural homology to the pore-forming proteins perfringolysin O and α-hemolysin. CofJ binds to lipid vesicles and epithelial cells, suggesting a role in membrane attachment during ETEC colonization.