Key points Paramyotonia congenita is a hereditary channelopathy caused by missense mutations in the SCN4A gene, which encodes the α subunit of the human skeletal muscle voltage‐gated sodium channel NaV1.4. Affected individuals suffered from myotonia and paralysis of muscles, which were aggravated by exposure to cold. We report a three‐generation Chinese family with patients presenting paramyotonia congenita and identify a novel N1366S mutation of NaV1.4. Whole‐cell electrophysiological recordings of the N1366S channel reveal a gain‐of‐function change of gating in response to cold. Modelling and molecular dynamic simulation data suggest that an arginine‐to‐serine substitution at position 1366 increases the distance from N1366 to R1454 and disrupts the hydrogen bond formed between them at low temperature. We demonstrate that N1366S is a disease‐causing mutation and that the temperature‐sensitive alteration of N1366S channel activity may be responsible for the pronounced paramyotonia congenita symptoms of these patients. Abstract Paramyotonia congenita is an autosomal dominant skeletal muscle channelopathy caused by missense mutations in SCN4A, the gene encoding the α subunit of the human skeletal muscle voltage‐gated sodium channel NaV1.4. We report a three‐generation family in which six members present clinical symptoms of paramyotonia congenita characterized by a marked worsening of myotonia by cold and by the presence of clear episodes of paralysis. We identified a novel mutation in SCN4A (Asn1366Ser, N1366S) in all patients in the family but not in healthy relatives or in 500 normal control subjects. Functional analysis of the channel protein expressed in HEK293 cells by whole‐cell patch clamp recording revealed that the N1366S mutation led to significant alterations in the gating process of the NaV1.4 channel. The N1366S mutant displayed a cold‐induced hyperpolarizing shift in the voltage dependence of activation and a depolarizing shift in fast inactivation, as well as a reduced rate of fast inactivation and accelerated recovery from fast inactivation. In addition, homology modelling and molecular dynamic simulation of N1366S and wild‐type NaV1.4 channels indicated that the arginine‐to‐serine substitution disrupted the hydrogen bond formed between N1366 and R1454. Together, our results suggest that N1366S is a gain‐of‐function mutation of NaV1.4 at low temperature and the mutation may be responsible for the clinical symptoms of paramyotonia congenita in the affected family and constitute a basis for studies into its pathogenesis.