Pannexin 1 (PANX1) is a glycoprotein that forms large pore channels capable of passing ions and metabolites such as ATP for cellular communication. PANX1 has been implicated in many diseases including breast cancer and melanoma, where inhibition or deletion of PANX1 reduced the tumorigenic and metastatic properties of the cancer cells. We interrogated the effect of single amino acid changes in various PANX1 domains using naturally occurring variants reported in cancer patient tumors. We found that a previously reported variant (Q5H) is present in cancer cells, but was not different from the wild type (Q5) in glycosylation, trafficking, or channel function and did not affect cellular properties. We discovered that the Q5H variant is in fact the highly conserved ancestral allele of PANX1 with 89% of humans carrying at least one Q5H allele. Another mutated form Y150F, found in a melanoma patient tumor, prevented phosphorylation at Y150 as well as complex N-glycosylation while increasing intracellular localization. Sarcoma (SRC) is the predicted kinase to phosphorylate the Y150 residue, and its phosphorylation is not likely to be constitutive, but rather dynamically regulated. The Y150 phosphorylation site is the first one reported to play a role in regulating posttranslational modifications and trafficking of PANX1, with potential consequences on its large-pore channel structure and function in melanoma cells.