[Objective] Heterologous expression of some human genes in yeast cells leads a severe growth defect. This phenomenon can occur when intrinsic yeast regulatory mechanisms are perturbed by the physiological function of the ectopically expressed human protein. Yeast cells are amenable to genetic studies and used as a platform for high-throughput screening. Thus, yeast cells harboring such growth inhibitory human proteins may be useful to analyze physiological function of the human protein and applied to find its inhibitor. In the present study, we constructed a collection of yeast expression plasmid harboring human protein-coding genes. Using the human gene library, we established a screening system to identify genes whose expression cause a growth defect in yeast cells. [Methods] Using the Gateway recombination technology, human protein-cording genes were cloned into a yeast expression plasmid. The resulting plasmids were individually transformed into yeast cells and analyzed whether their expression effects on yeast growth. Furthermore, identified inhibitory genes were expressed as GFP fusion proteins and their localization in yeast cells were analyzed. [Results and Conclusion] Among 2991 human protein-cording genes, we identified 29 genes whose expression caused a sever growth defect in yeast cells. Some of them are causative genes to develop human disorders. For example, PDLIM4 is involved in development of osteoporosis and prostate cancer. Physiological function of PDLIM4 has not been understand very well. Our study may provide another option to investigate human proteins.