Pseudogenes are classically thought of as nonfunctional DNA sequences due to their inability to be translated, or to produce a functional protein. Gap junctions, a multiprotein complex made of proteins called connexins, are involved in intercellular communication and are deregulated in many cancers. Connexin43 (Cx43) is the only connexin for which a pseudogene has been reported so far. The Cx43 pseudogene (PsiCx43) has all of the features of an expressed gene. We identified the presence of a PsiCx43 mRNA transcript in several cancer cell lines and in none of the normal mammary epithelial cells studied. Using an in vitro translation assay, we found that the PsiCx43 coding plasmid could be translated into a 43 kDa protein. This was further confirmed by expressing a PsiCx43-green fluorescence protein fusion protein in breast cancer MCF-7 cells. We then examined the functional significance of the PsiCx43. In both MTT growth and colony formation assays, significant growth inhibition was observed, a feature common to cells overexpressing the Cx43 gene. However, using a scrape-loading assay, we could not detect any effect on gap junctional intercellular communication. Based on our findings, PsiCx43 joins and enlarges the thus far restricted group of functionally transcribed and translated pseudogenes.