The irritancy of topical products has to be investigated to ensure the safety and compliance. Although several reconstructed human epidermal models have been adopted by the Organization for Economic Cooperation and Development (OECD) to replace in vivo animal irritation testing, these models are based on a single cell type and lack dermal components, which may be insufficient to reflect all of the components of irritation. In our study, we investigated the use of acellular porcine peritoneum extracellular matrix as a substrate to construct full-thickness human skin equivalents (HSEs) for use as irritation screening tool. The acellular peritoneum matrix (APM) exhibited excellent skin cell attachment (>80%) and proliferation for human dermal fibroblasts (HDF) and immortalized human keratinocytes (HaCaT). APM-HSEs based on coculture of HDF and HaCaT were prepared. Increased HDF seeding density up to 5 × 10(4)/cm(2) resulted in APM-HSEs with a thicker and more organized epidermis. The epidermis of APM-HSEs expressed keratin 15, a keratinocyte proliferation marker, and involucrin, a differentiation marker, respectively. To assess the use of APM-HSEs for irritation testing, six proficiency chemicals, including three nonirritants (phosphate-buffered saline, polyethylene glycol 400, and isopropanol) and three irritants (1-bromohexane, heptanol, and sodium dodecyl sulfate) were applied. The APM-HSEs were able to discriminate nonirritants from irritants based on the viability. Levels of cytokines (interleukin [IL]-1α, IL-1ra, IL-6, IL-8, and granulocyte macrophage colony-stimulating factor [GM-CSF]) in these treatment groups further assisted the irritancy ranking. In conclusion, we have developed partially differentiated full-thickness APM-HSEs based on acellular porcine peritoneum matrix, and these APM-HSEs demonstrated utility as an in vitro irritation screening tool.