The extraction of important features in cancer cell image analysis is a key process in grading renal cell carcinoma. In this study, we applied three-dimensional (3D) texture feature extraction methods to cell nuclei images and evaluated the validity of them for computerized cell nuclei grading. Individual images of 2,423 cell nuclei were extracted from 80 renal cell carcinomas (RCCs) using confocal laser scanning microscopy (CLSM). First, we applied the 3D texture mapping method to render the volume of entire tissue sections. Then, we determined the chromatin texture quantitatively by calculating 3D gray-level co-occurrence matrices (3D GLCM) and 3D run length matrices (3D GLRLM). Finally, to demonstrate the suitability of 3D texture features for grading, we performed a discriminant analysis. In addition, we conducted a principal component analysis to obtain optimized texture features. Automatic grading of cell nuclei using 3D texture features had an accuracy of 78.30%. Combining 3D textural and 3D morphological features improved the accuracy to 82.19%. As a comparative study, we also performed a stepwise feature selection. Using the 4 optimized features, we could obtain more improved accuracy of 84.32%. Three dimensional texture features have potential for use as fundamental elements in developing a new nuclear grading system with accurate diagnosis and predicting prognosis.