Transmembrane protein 166 (TMEM166) is a novel human regulator involved in both autophagy and apoptosis. In this study, we generated a specific rabbit polyclonal antibody against human TMEM166 and assessed the expression of this protein in various human normal and tumor tissue samples by tissue microarray-based immunohistochemical analysis. Varying TMEM166 protein levels were expressed in a cell-type and tissue-type-specific manner in detected tissues or organs. Strong TMEM166 expression was shown in the glomerular zona of the adrenal cortex, chromophil cells of the pituitary gland, islet cells, squamous epithelium of the esophagus mucosa, the fundic gland, and hepatocytes. Moderate or weak TMEM166 staining was identified in the parathyroid gland, the testis, vaginal stratified squamous cells, lung macrophages, hematopoietic cells, renal tubular epithelial cells, macrophages in the spleen red pulp, and neuronal cells in the cerebral cortex. Some tissues failed to stain for TMEM166, such as adipose tissue, colon, cerebellum, lymph node, mammary gland, ovary, prostate, rectum, skin, small intestine, thyroid gland, tonsil, and thymus. In comparing human normal and tumor tissues, TMEM166 expression was widely downregulated in the cancer tissues. Our studies provide the basis for future investigations into cell-type-specific functions of this protein in human normal and tumor tissues.