The mitral valve prolapse (MVP) is a common cardiac disorder which affects 2-4% of the population and remains one of the most frequent indications for valvular surgery. The familial nature of MVP has been proposed for many years and so far, FLNA remains the only identified gene. Recently, it has been shown that FLNA mutations deregulate the RhoA/ Rac1 GTPases balance and provided evidences for a role of the Rac1 specific GTPase activating protein, FilGAP, in this network. FilGAP is a recognized FlnA-binding RhoGTPase-activating protein. Giving the tight interactions of FlnA and FilGAP, we first tested, using a candidate gene approach, the hypothesis that FilGAP, encoded by ARHGAP24, could be involved in MVP. We have sequenced ARHGAP24 in 95 MVP operated patients and identified 3 rare missense mutations in highly conserve residues (FilGAP p.R95Q; p.P417H and p.T481M). One mutation was novel and the 2 others present a minor allele frequency lower than 0.1% in EVS. Moreover, p.T481M co-segregates with the pathology in a family with 3 affected patients. We then investigated the impact of these mutations in HEK293 cells. The role of FilGAP is to decrease Rac1 activity and thus to regulate cell processes involved in actin cytoskeleton properties as adhesion, protrusion and intracellular dynamics. From pull-down assays, we have shown that FilGAP mutations alter Rac1 GTPase activity and significantly decrease the FilGAP interaction with the active form of Rac1 (p<0.01). We have also shown, using the XCELLigence system, that cell adhesion and spreading was significantly increased with mutated FilGAP (p<0.01). Our results indicate that ARHGAP24 variants are loss-function mutations. Moreover, we demonstrate that FilGAP mutations alter the downstream signaling pathway by two different mechanisms. FilGAP p.P417H and p.T481M decrease the interaction with FlnA while p.R95Q impacts the plasma membrane anchorage. This work reinforces the involvement of GTPases pathway in MVP pathogenesis.