Podocytes are highly specialized epithelial cells located at the outer aspects of the glomerular capillary tuft and critical components of the kidney filtration barrier. To maintain their unique features podocytes express a number of proteins that are only sparsely found elsewhere in the body. In this study, we have identified four (Tmem234, Znf185, Lrrc49, and Slfn5) new highly podocyte-enriched proteins. The proteins are strongly expressed by podocytes while other parts of the kidney show only weak or no expression. Tmem234, Slfn5 and Lrrc49 are located to foot processes, whereas Znf185 is found in both foot and major processes. Expressional studies in developing kidneys show that these proteins are first expressed at the capillary stage glomerulus, the same stage when the formation of major and foot processes begins. We identified zebrafish orthologues for Tmem234 and Znf185 genes, and inactivated their expression using morpholino technology. Studies in zebrafish larvae indicate that Tmem234 is essential for the organization and functional integrity of the pronephros filtration barrier as knockdown of Tmem234 expression results in foot process effacement and proteinuria. In summary, we have identified five novel highly podocyte-enriched proteins and show that one of them, Tmem234, is essential for the normal filtration barrier in zebrafish pronephros. Identification of new molecular components of the kidney filtration barrier opens up possibilities to study their role in the glomerulus biology and diseases.