Gap junctions (GJs), which consist of proteins called connexins, are intercellular channels that allow the passage of ions, second messengers, and small molecules. GJs and connexins are considered as emerging therapeutic targets for various diseases. Previously, we screened numerous compounds using our recently developed iodide yellow fluorescent protein gap junctional intercellular communication (I-YFP GJIC) assay and found that flunarizine (FNZ), used for migraine prophylaxis and as an add-on therapy for epilepsy, inhibits GJIC in LN215 human glioma cells. In this study, we confirmed that FNZ inhibits GJIC using the I-YFP GJIC assay. We demonstrated that FNZ inhibits GJ activities via a mechanism that is independent of calcium channels and dopaminergic D2, histaminergic H1, or 5-HT receptors. In addition, we showed that FNZ significantly increases connexin 43 (Cx43) phosphorylation on the cell surface, but does not alter the total amount of Cx43. The beneficial effects of FNZ on migraines and epilepsy might be related to GJ inhibition.