Background We have recently shown that the expression of nestin, a progenitor/stem cell marker protein, is localized in different mesenchymal compartments in human skin including the sweat gland stroma. Objectives As other exocrine glands are recognized sources of multipotent stem cell populations with potential for multilineage differentiation, it was our aim to isolate, expand and characterize glandular stem cells from human sweat glands. Methods Isolation of human sweat glands was based on mechanical and enzymatic digestion of axillary skin. Cultivation was performed on collagen-coated cell culture dishes and the resulting cell population was investigated at the protein and mRNA level. Results Outgrowing cells of isolated sweat glands showed a high-proliferation activity and were characterized by nestin expression in more than 80% of the cells. These sweat gland stem cells could be maintained in culture for long periods of time and showed spontaneous differentiation into cells representative of the different germ layers. Conclusions This pilot study provides the first, simple protocol for the isolation of adult human nestin-positive stem cells from the sweat gland mesenchyme, which promises to provide an easily accessible and abundantly available, autologous source of multipotent stem cells for cell-based regenerative medicine applications.