Mutations in the bestrophin-1 (Best1) gene are linked to several kinds of macular degeneration in both humans and dogs. Although bestrophins have been shown clearly to be Cl(-) ion channels, it is controversial whether Cl(-) channel dysfunction can explain the diseases. It has been suggested that bestrophins are multifunctional proteins: they may regulate voltage-gated Ca(2+) channels in addition to functioning as Cl(-) channels. Here, we show that human Best1 gene (hBest1) differentially modulates Ca(V)1.3 (L-type) voltage-gated Ca(2+) channels through association with the Ca(V)beta subunit. In transfected human embryonic kidney 293 cells, hBest1 inhibited Ca(V)1.3. Inhibition of Ca(V)1.3 was not observed in the absence of the beta subunit. Also, the hBest1 C terminus binds to Ca(V)beta subunits, suggesting that the effect of hBest1 was mediated by the Ca(V)beta subunit. The region of hBest1 responsible for the effect was localized to a region (amino acids 330-370) in the cytoplasmic C terminus that contains a predicted src-homology-binding domain that is not present in other bestrophin subtypes. Mutation of Pro(330) and Pro(334) abolished the effects of hBest1 on Ca(V)1.3. The effect was specific to hBest1; it was not observed with mouse Best1 (mBest1), mBest2, or mBest3. Wild-type hBest1 and the disease-causing mutants R92S, G299R, and D312N inhibited Ca(V) currents the same amount, whereas the A146K and G222E mutants were less effective. We propose that hBest1 regulates Ca(V) channels by interacting with the Ca(V)beta subunit and altering channel availability. Our findings reveal a novel function of bestrophin in regulation of Ca(V) channels and suggest a possible mechanism for the role of hBest1 in macular degeneration.