Regulatory T cells (Tregs), identified by the expression of CD4, CD25 and Foxp3, together with immunosuppressive macrophages, such as CD163+ M2 macrophages, are involved in maintaining peripheral tolerance. The aim of this study was to elucidate the involvement of Tregs and CD163+ macrophages in invasive and non-invasive extramammary Paget's disease. The presence of CD4+CD25+Foxp3+ Tregs, CD163+ M2 macrophages and matrix metalloproteinase-9+ cells was examined immunohistologically in fixed sections of lesional skin from 10 patients with non-invasive extramammary Paget's disease and 7 patients with invasive extramammary Paget's disease. Fewer CD4+CD25+Foxp3+ Tregs were observed in non-invasive extramammary Paget's disease than in invasive extramammary Paget's disease. In contrast, higher numbers of CD163+ macrophages and metalloproteinase-9+ cells were detected only in invasive extramammary Paget's disease. These findings suggest that the induction of immunosuppressive cells in extramammary Paget's disease differs according to the tumour stage.