Dysregulation of Connexin (CX) expression and function is associated with a range of chronic inflammatory conditions including psoriasis and nonhealing wounds. To mimic a proinflammatory environment, HaCaT cells, a model human keratinocyte cell line, were challenged with 10 µg/ml peptidoglycan (PGN) isolated from Staphylococcus aureus for 15 min to 24 hr in the presence or absence of CX blockers and/or following CX26, CX43, PANX1 and TLR2 small interfering RNA (siRNA) knockdown (KD). Expression levels of IL‐6, IL‐8, CX26, CX43, PANX1, TLR2 and Ki67 were assessed by quantitative real‐time polymerase chain reaction, western blot analysis and/or immunocytochemistry. Nuclear factor kappa β (NF‐κβ) was blocked with BAY 11‐7082, CX‐channel function was determined by adenosine 5′‐triphosphate (ATP) release assays. Enzyme‐linked immunosorbent assay monitored IL6 release following PGN challenge in the presence or absence of siRNA or blockers of CX or purinergic signalling. Exposure to PGN induced IL‐6, IL‐8, CX26 and TLR2 gene expression but it did not influence CX43, PANX1 or Ki67 messenger RNA expression levels. CX43 protein levels were reduced following 24 hr PGN exposure. PGN‐induced CX26 and IL‐6 expression were also aborted by TLR2‐KD and inhibition of NF‐κβ. ATP and IL‐6 release were stimulated following 15 min and 1–24 hr challenge with PGN, respectively. Release of both agents was inhibited by coincubation with CX‐channel blockers, CX26‐, CX43‐ and TLR2‐KD. The IL‐6 response was also reduced by purinergic blockers. CX‐signalling plays a role in the innate immune response in the epidermis. PGN is detected by TLR2, which via NF‐κβ, directly activates CX26 and IL‐6 expression. CX43 and CX26 maintain proinflammatory signalling by permitting ATP release, however, PANX1 does not participate.