AbstractPannexin (PANX) channels are present in skin and facilitate the movement of signalling molecules during cellular communication. PANX1 and PANX3 function in skin homeostasis and keratinocyte differentiation but were previously reduced in a small cohort of human cutaneous squamous cell carcinoma (cSCC) tumours compared to normal epidermis. In our study we used SCC‐13 cells, limited publicly available RNA‐seq data and a larger cohort of cSCC patient‐matched samples to analyse PANX1 and PANX3 expression and determine the association between their dysregulation and the malignant properties of cSCC. In a bioinformatics analysis, PANX1 transcripts were increased in cSCC and head and neck SCC tumours compared to normal tissues, but PANX3 mRNA showed no differences. However, in our own cohort PANX3 transcripts were decreased in cSCC compared to patient‐matched aged skin, whereas PANX1 protein was upregulated in cSCC. PANX1 localized to all regions within the cSCC tumour microenvironment, and increased levels were associated with larger tumour dimensions. To investigate PANX1 function in SCC‐13 cells, we deleted PANX1 via CRISPR/Cas9 and treated with PANX1 inhibitors, which markedly reduced cell growth and migration. To assess PANX3 function in cutaneous carcinogenesis, we employed the 7,12‐dimethylbenz(a)anthracene/12‐otetradecanoylphorbol‐13‐acetate (DMBA/TPA) model using our global Panx3 knockout (KO) mice, where 60% of wild‐type and 100% of KO mice formed precancerous papillomas. Average papilloma volumes at endpoint were significantly increased in KO mice and showed moderate evidence of increases in KO mice over time. Collectively, these findings suggest PANX1 and PANX3 dysregulation may have potential tumour‐promoting and tumour‐suppressive effects for keratinocyte transformation, respectively. imageKey points Pannexin 1 (PANX1) and pannexin 3 (PANX3) are channel‐forming proteins which are critical in the normal maintenance and function of keratinocytes in the skin but may become altered in cutaneous squamous cell carcinoma (cSCC) tumours. In this study we used a combination of culture models, mouse models and patient‐derived tissues. We found PANX1 levels are increased in cSCC tumours and present in all tumour regions, functioning to promote cSCC cell growth and migration. Conversely, PANX3 levels are decreased in cSCC tumours, and this protein reduces the incidence and growth of precancerous lesions. Taken together our data indicate that in cSCC these pannexin family members seem to have opposite effects, in either promoting or restricting cancer cell properties. These results help us to better understand the mechanisms of malignant transformation of keratinocytes and offer a new potential therapeutic target for the treatment of advanced cSCC.