Mutations in Connexin26 (Cx26) give rise to a spectrum of dominantly inherited hyperproliferating skin disorders, the severest being keratitis-ichthyosis-deafness (KID) syndrome, an inflammatory skin disorder, with patients prone to opportunistic infections. We compared the effects of peptidoglycan (PGN) extracted from the skin commensal Staphylococcus epidermidis and the opportunistic pathogen Staphylococcus aureus on interleukin-6 and connexin expression in HaCaT cells (a keratinocyte cell line) and connexin channel activity in HaCaT and HeLa (connexin deficient) cells transfected to express KID and non-KID Cx26 mutations. In both cell types, PGN from S. aureus induced hemichannel activity in cells expressing KID mutants as monitored by ATP release assays following 15-min challenge, while that from S. epidermidis evoked a response in HeLa cells. In KID mutant expressing cells, ATP release was significantly higher than in cells transfected with wild-type Cx26. No ATP release was observed in non-KID mutant transfected cells or in the presence of carbenoxolone, a connexin channel blocker. PGN isolated from S. aureus but not S. epidermidis induced interleukin-6 and Cx26 expression in HaCaT cells following 6-h challenge. Challenge by PGN from S. aureus evoked a greater interleukin-6 response in cells expressing KID mutants than in cells expressing wtCx26 or non-KID mutants. This response returned to basal levels if acute KID hemichannel signalling was blocked prior to PGN challenge. Thus, KID mutants form channels that can be triggered by the pro-inflammatory mediator PGN from opportunistic pathogens but not skin commensals, providing further insight into the genotype-phenotype relationship of Cx26 disorders.