Connexin43 (Cx43; gene name GJA1) is the most ubiquitously expressed gap junction protein, and understanding of its regulation largely falls under transcription and post-translational modification. In addition to Cx43, Gja1 mRNA encodes internally translated isoforms regulating gap junction formation, whose expression is modulated by TGF-β. Here, using RLM-RACE, we identify distinct Gja1 transcripts differing only in 5′ UTR length, of which two are upregulated during TGF-β exposure and hypoxia. Introduction of these transcripts into Gja1−/− cells phenocopies the response of Gja1 to TGF-β with reduced internal translation initiation. Inhibiting pathways downstream of TGF-β selectively regulates levels of Gja1 transcript isoforms and translation products. Reporter assays reveal enhanced translation of full-length Cx43 from shorter Gja1 5′ UTR isoforms. We also observe a correlation among UTR selection, translation, and reduced gap junction formation in aged heart tissue. These data elucidate a relationship between transcript isoform expression and translation initiation regulating intercellular communication.