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 erythrokeratodermia variabilis(EKV) ， hearing impairment and peripheral neuropathy. The pathological mechanism for Cx31 mutants in these diseases remains unknown. Assembly, intracellular transport, plaque assembly and stability and channel conductivity of Cx31 are finely regulated and likely involve proteins that interact with Cx31. However, little is known about the Cx31 interaction proteins. And then the differential immunogenic peptide was synthesized, the purified peptide was coupled to the hemocyanin from keyhole limpets(KLH). The peptide-KLH solution was mixed with freund's complete or incomplete adjuvant and immunized rabbit to prepare specific polyclonal antibody against the topology of connexin31. A proteomics approach was used to screen Cx31 binding proteins using HT1080 cells stably expressing a myc-tagged Cx31. Immunoprecipitation followed by peptide sequence analysis identified association of actin and annexin Ⅱ . Interaction between annexin Ⅱ and Cx31 is further confirmed by coimmunoprecipitation, and immuno-colocalization. This result would be useful to study the function of connexin31 further.