KIBRA has been described as a key regulator of the Hippo signaling pathway, regulating organ size control, cell contact inhibition, cell growth, as well as tumorigenesis and cystogenesis. Since there is scarce information on KIBRA gene expression regulation, we analyzed the molecular basis of tissue-specific KIBRA expression in human kidney epithelial (IHKE, HPCT) and neuroblastoma (SH-SY5Y, SK-SN-SH) cells. We detected four novel and differentially used transcription start sites, two of which positioned in the first intron, generating two novel alternative exons. We identified one constitutively active core promoter (P1a) and three alternative promoters (P1b, P2, and P3), which were exclusively active in kidney cells. Transcription factor 7-like 2 (TCF7L2) selectively activated KIBRA at P1a, P2, and P3 in kidney cells. The two genetic variants -580C>T (p < 0.05) and -1691C>T (p < 0.01) significantly affected the transcriptional activity of the KIBRA core promoter. We propose a novel functional structure of the KIBRA gene and provide detailed insight into molecular cell type-specific KIBRA transcriptional regulation by TCF7L2, the Yes-associated protein 1 and TEA domain family member. Our findings provide a potential basis for future studies on malfunctioning KIBRA regulation in pathophysiological conditions such as cancer development. KEY MESSAGE: KIBRA expression is regulated by three independent, cell type-specific promoters Two novel TSS were located within intron one resulting in two alternative exons TSS utilization is cell type-specific TCF7L2, YAP1, and TEAD are involved in the differential KIBRA expression regulation.