Precise regulation of calcium homeostasis is essential for many physiological functions. The Ca²⁺-selective transient receptor potential (TRP) channels TRPV5 and TRPV6 play vital roles in calcium homeostasis as Ca²⁺ uptake channels in epithelial tissues. Detailed structural bases for their assembly and Ca²⁺ permeation remain obscure. Here we report the crystal structure of rat TRPV6 at 3.25 Å resolution. The overall architecture of TRPV6 reveals shared and unique features compared with other TRP channels. Intracellular domains engage in extensive interactions to form an intracellular ‘skirt’ involved in allosteric modulation. In the K⁺ channel-like transmembrane domain, Ca²⁺ selectivity is determined by direct coordination of Ca²⁺ by a ring of aspartate side chains in the selectivity filter. On the basis of crystallographically identified cation-binding sites at the pore axis and extracellular vestibule, we propose a Ca²⁺ permeation mechanism. Our results provide a structural foundation for understanding the regulation of epithelial Ca²⁺ uptake and its role in pathophysiology.