Purpose: We have previously reported that skin barrier function is disrupted in mice with colonic tumours induced by azoxymethane (AOM) and dextran sodium sulphate (DSS). We postulated that the impaired skin barrier function was associated with reactive oxygen species derived from gp91phox. In this study, we investigated the mechanisms underlying the impaired skin barrier function using gp91phox−/− mice. Materials and methods: We induced colonic tumorigenesis in C57BL/6j mice by AOM + DSS administration and evaluated the influence of reactive oxygen species on skin barrier function by using the hydroxyl radical scavenger N-acetyl-l-cysteine (NAC) or gp91phox−/− mice. Damage to the colon and skin following treatment with AOM + DSS was monitored using protein analysis methods and by detection of inflammatory mediators in the plasma. Results: NAC could not prevent the increase in transepidermal water loss (TEWL) and decrease in skin hydration level caused by AOM + DSS in gp91phox+/+ mice. However, gp91phox−/− mice showed no change in TEWL and skin hydration level. The dermal expression levels of nucleotide-binding domain, leucine-rich containing family, pyrin-domain containing 3 (NLRP3), and caspase-1 were reduced in gp91phox−/− mice. Moreover, the plasma concentrations of interleukin-18 and thymic stromal lymphopoietin (TSLP) were lower in gp91phox−/− mice than those in gp91phox+/+ mice. Inhibition of hydrogen peroxide production from superoxide anions in the gp91phox−/− status prevented the increased TEWL and decreased skin hydration level noted with degradation of NLRP3 and caspase-1. Conclusions: Superoxide anions may play an important role in the onset of the impaired skin barrier function in mice with colonic tumours.