Heart valve diseases affect 3% of world population, and surgery is often the only therapeutic mean. A genetic study performed on a family in which several members exhibited a mitral valve prolapse (MVP) identified a mutation on LRRFip1 gene. LRRFip1 undergoes extensive alternative transcription splicing giving rise to five isoforms in humans and the mutation we identified results in R94G substitution in three (Iso1, 3 and 4) out of the five isoforms. Previous studies essentially focused on LRRFip1-Iso5 that was first described as a transcription factor interacting with positive (Dishevelled) and negative (Flightess-1) regulators of the canonical Wnt b-catenin dependent pathway. LRRFip1 thus appeared as an interesting gene in MVP as it may participate and regulate two crucial events of cardiac valve development and homeostasis involving Wnt pathway: epithelial to mesenchymal transition and cell proliferation. Interestingly, we showed using RNA sequencing analysis and RTPCR experiments that LRRFip1-Iso1 is the most expressed isoform in human valves. Furthermore, LRRFip1-Iso1 has poor homology with the other isoforms and nothing is known about its function. We thus focused on LRRFIP1-Iso1 and analyzed its cellular localization, its role in Wnt pathway and the impact of R94G mutation in HEK293 cells. Cell fractionation experiments revealed a nuclear localization of LRRFip1-Iso1 while other isoforms are strictly cytoplasmic. We then showed by Luciferase assays and co-immunoprecipitations that out of the five isoforms, LRRFip1-Iso1 activates the canonical Wnt pathway at the highest levels and interacts mainly with Fli-1. Furthermore, R94G mutation decreases this interaction. Thus, our studies suggest that LRRFip1-Iso1 may activate the canonical Wnt pathway by opposing Fli-1 inhibitory effects. R94G mutation may alter this key regulatory loop, deregulate gene transcription and consequently alter valvulogenesis and/or valve homeostasy.