The primary cilium is a paradigmatic subcellular compartment at the nexus of numerous cellular and morphogenetic pathways. The tubby family protein TULP3 acts as an adapter of the intraflagellar transport complex A in transporting integral membrane and membrane-associated lipidated proteins into cilia. However, the mechanisms by which TULP3 coordinates ciliary transport of diverse cargoes is not well understood. Here, we provide molecular insights into TULP3-mediated ciliary cargo recognition. We screened for critical TULP3 residues by proximity biotinylation-mass spectrometry, structural analysis, and testing TULP3 variants in human patients with hepatorenal fibrocystic disease and spina bifida. The TULP3 residues we identified 1) were located on one side of the β-barrel of the tubby domain away from the phosphoinositide binding site, 2) mediated ciliary trafficking of lipidated and transmembrane cargoes, and 3) determined proximity with these cargoes in vivo without affecting ciliary localization, phosphoinositide binding or hydrodynamic properties of TULP3. Overall, these findings implicate a specific region of one of the surfaces of the TULP3 β-barrel in ciliary trafficking of diverse cargoes. This region overlooks the β-strands 8-12 of the β-barrel and is away from the membrane anchoring phosphoinositide binding site. Targeting the TULP3-cargo interactions could provide therapeutics in ciliary trafficking diseases.