Although various samples, including tissue, cells, serum, and urine, from patients with renal cell carcinoma (RCC) have been analyzed, biomarkers with diagnostic value have yet to be identified. We used a proteomics approach to analyze cyst fluid in cases of cyst-associated RCC to identify accessible and abundant proteins that are overexpressed and/or secreted by RCC cells. Proteins in the cyst fluid were separated by reverse-phase high-performance liquid chromatography and agarose two-dimensional gel electrophoresis and were identified by tandem mass spectrometry. We conducted a National Center for Biotechnology Information search and a MEDLINE search to predict the function of these identified proteins and to select a tumor-marker candidate protein. Our search resulted in the identification and selection of the differentially regulated protein known as 14-3-3 protein beta/alpha, which was overexpressed in cyst fluid from cyst-associated RCC but has not been previously associated with RCC. We then measured its incidence through Western blotting of various normal and RCC samples (serum, urine, tissue, and cyst fluid). The expression levels of 14-3-3 protein beta/alpha were higher in urine samples from patients with RCC than in samples from healthy volunteers. Receiver operating characteristic (ROC) curve analyses were performed to assess this potential biomarker; these data (area under the ROC curve value was 0.8813) indicate a high degree of accuracy for this screening method. 14-3-3 Protein beta/alpha may be a diagnostically useful biomarker for early diagnosis of RCC.