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. A proteomics approach was applied 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 Cx31. Interaction between actin and Cx31 is further confirmed by coimmunoprecipitation and immuno-colocalization. Pharmacological disruption of actin polymerization inhibits the plasma membrane localization of Cx31. The results suggested the actin cytoskeleton may play an essential role in regulating Cx31 trafficking via direct association.