Abstract Gap junctions formed from connexins (Cx) have been reported to regulate proliferation, differentiation and apoptosis through formation of intercellular channels between opposing cells. Interestingly, connexins have been identified as tumor suppressors and enhancers, a distinction dependent on the type and stage of disease. In melanomas, the role of connexins during cancer onset and progression remains unclear. In the current study, we hypothesize that expression of Cx26 and Cx43 in BL6 mouse melanomas will increase gap junctional intercellular communication (GJIC) thereby decrease their tumorigenic properties. Western blots, immunofluorescence and dye coupling studies revealed that BL6 cells express low levels of Cx26 , Cx43 and are poorly coupled. When cells were engineered to express Cx26 or Cx43, dye transfer studies revealed that both Cx26- and Cx43-expressing cells were highly coupled. Interestingly, when these connexin-rich melanomas were co-cultured with Cx43-rich keratinocytes, melanoma microtumors remained functionally isolated and failed to couple to surrounding keratinocytes. Growth curve studies revealed that exogenous Cx43, but not Cx26, significantly reduced total cell numbers; that was primarily due to reduced cell proliferation. Furthermore, Cx43 expression significantly reduced anchorage-independent growth compared to controls, however, migration remained unaffected. Additionally, Cx43-expressing melanoma cells displayed a reduction in growth when co-cultured with keratinocytes, despite a lack of heterocellular GJIC. Finally, when grown in in vivo avian embryo model, primary tumor weight was reduced in tumor cells expressing Cx43 , but not in Cx26-expressing melanomas , as compared to controls. Overall, our results would suggest that Cx43 is a potent tumor suppressor in melanomas. Supported by the Canadian Institute of Health Research to DWL. Citation Format: Qing C. Shao, Mark Jake Ableser, Silvia Penuela, Dale W. Laird. Connexin43 reduces melanoma growth within a keratinocyte microenvironment and during tumorigenesis in vivo. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2467. doi:10.1158/1538-7445.AM2014-2467