Patients with high-risk long QT syndrome (LQTS) mutations may experience life-threatening cardiac events. The present study sought to characterize a novel pathogenic mutation, KCNQ1p.Thr312del, in a Chinese LQT1 family. Clinical and genetic analyses were performed to identify this novel causative gene mutation in this LQTS family. Autosomal dominant inheritance of KCNQ1p.T312del was demonstrated in the three-generation pedigree. All mutation carriers presented with prolonged QT intervals and experienced recurrent syncope during exercise or emotional stress. The functional consequences of the mutant channel were investigated by computer homology modeling as well as whole-cell patch-clamp, western-blot and co-immunoprecipitation techniques using transfected mammalian cells. T312 is in the selectivity filter (SF) of the pore region of the KCNQ1-encoded channel. Homology modeling suggested that secondary structure was altered in the mutant SF compared with the wild-type (WT) SF. There were no significant differences in K_v7.1 expression, membrane trafficking or physical interactions with KCNE1-encoded subunits between the WT and mutant transfected channels. However, the KCNQ1p.T312del channels expressed in transfected cells were non-functional in the absence or presence of auxiliary KCNE1-subunits. Dominant-negative suppression of current density and decelerated activation kinetics were observed in cells expressing KCNQ1WT and KCNQ1p.T312del combined with KCNE1 (KCNQ1WT/p.T312del + KCNE1 channels). Those electrophysiological characteristics underlie the pathogenesis of this novel mutation and also suggest a high risk of cardiac events in patients carrying KCNQ1p.T312del. Although protein kinase A-dependent current increase was preserved, a significant suppression of rate-dependent current facilitation was noted in the KCNQ1WT/p.T312del + KCNE1 channels compared to the WT channels during 1- and 2-Hz stimulation, which was consistent with the patients’ phenotype being triggered by exercise. Overall, KCNQ1p.Thr312del induces a loss of function in channel electrophysiology, and it is a high-risk mutation responsible for LQT1.