Connexins form gap junctions that mediate the transfer of ions, metabolites, and second messengers between contacting cells. Connexin31(Cx31) is an important member of connexin β family. Mutations in Cx31 are associated with erythrokeratoderma variabilis (EKV) ， hearing impairment and peripheral neuropathy. The pathological mechanism for Cx31 mutants in these diseases remains unknown. Using Site-directed mutagenesis (SDM) to construct two dominant hearing impairment associated Cx31 mutant expression plasmids, R180X-pEGFP-N1 and E183K- pEGFP-N1. A number of functional analysis were described to investigate the effect of two dominant hearing impairment-associated Cx31 mutants, R180X and E183K, on connexon trafficking. The majority of the mutant R180X and E183K were cytoplasmic, they can not traffic to normal intracellular localization and can not form functional gap junction channels. But not observed with wild-type Cx31.This may provide an insight into the pathological mechanism of Cx31 mutants.