Objective：To separate human kidney phosphoproteome by two-dimensional gel electrophoresis. Methods： The phosphorylated proteins from human kidney tissues were enriched with phosphate metal affinity chromatography （PMAC） resin. After being concentrated and desalted, the samples were separated by isoelectric focusing on first dimension and SDS electrophoresis on second dimension. Results： The phosphorylated proteins were successfully extracted from human kidney tissues and were separated by two-dimensional gel electrophoresis. Conclusion.. Phosphoprotein enrichment technique combined with two-dimensional gel electrophoresis is an effective approach to study phosphoproteome, laying a foundation for further investigation of human kidney phosphoproteins