Background Timothy syndrome (TS) is a rare long-QT syndrome caused by CACNA1C mutations G406R in exon 8A (TS1) and G402S/G406R in exon 8 (TS2). Management of TS is a challenge and prognosis is poor. This study aimed to explore the inheritance pattern and mechanism of an I-Na blocker, mexiletine, to improve clinical manifestations in TS. Methods and Results A 2-year-old Chinese girl with a typical TS1 phenotype underwent candidate gene screening. Qualitative and quantitative cloning sequence and analyses for mosaicism were performed on family members. Therapeutic effects of mexiletine were evaluated using ECG and Holter monitoring. The electrophysiological effect of mexiletine was evaluated in a TS model using rabbit ventricular wedges. The proband with severe syndactyly and delayed language skills was identified harboring a G406R mutation in CACNA1C. Her baseline ECG showed markedly prolonged QTc, 2:1 AV block and macro-T wave alternans. G406R was absent in her mother but expressed in her father's oral mucosa, sperm, and white blood cells, indicating a mosaic carrier. Although asymptomatic, he exhibited mild QTc prolongation (470-490 ms) and syndactyly. Mexiletine shortened QTc from 584 to 515 ms, blunted QT-RR relationship, and abolished 2:1 AV block and T wave alternans in the girl. In in vitro studies, mexiletine inhibited late I-Na with IC50 of 17.6 +/- 1.9 mu mol/L and attenuated brady-dependent QT prolongation and reduced QT-RR slope in the TS model using BayK 8644. Conclusions Mexiletine shortened QTc, attenuated QT-RR slope, abolished 2:1 AV block and T wave alternans in a TS1 patient and TS model via inhibition of late I-Na.