The testis is the male gonad responsible for spermatogenesis and male hormone secretion. The complicated processes of spermatogenesis and steroidogenesis determine the complexity of protein expression control in the testis. In this study, the heterogeneity of human testis proteins was investigated using 2-dimensional gel electrophoresis. A total of 847 protein spots corresponding to 462 unique proteins were identified successfully by mass spectrometry. Notable heterogeneity was evidenced by the presence of more than 1 spot with different molecular weight and/or Isoelectric point values for each of 180 different proteins. Analysis of the detected peptides of these proteins indicated that this heterogeneity was partly the result of alternative splicing and/or proteolysis. SP_PIR_Keywords analysis suggested that alternative initiation sites and various forms of posttranslational modification may also contribute toward this heterogeneity. Using Pro-Q Diamond phosphostain, 68 spots representing 52 proteins were stained, confirming the presence of phosphorylated forms of these proteins in the human testis. These data were used to establish a proteome reference database, which can be accessed over the Internet (http://reprod.njmu.edu.cn/2d). This database provides an initial reference map of the human testis and serves as a useful resource for comparative proteomics studies of the human testis under normal and pathological states. The abundant protein heterogeneity observed in this study and further investigation of its biological significance will contribute toward understanding protein expression regulation in the human testis and will generate insight into the molecular mechanism of spermatogenesis.