We aimed to delineate the morphogenesis of aberrant nuclear features of urothelial carcinoma (UC) cells in association with cytokeratin (CK) expression patterns and cell proliferation activity. Correlation analysis of the nuclear area by morphometry and the expression patterns of CK5, CK20 and Ki-67 by triple immunofluorescence analysis was applied to 1699 cells from five low-grade and seven high-grade cases of UC. The majority of UC cells showed aberrant cellular differentiation represented by abnormal CK expression patterns of CK5(+)/CK20(+) (40.5%) or CK5(−)/CK20(+) (56.0%). CK5(+)/CK20(−) cells, a phenotype of cancer stem/progenitor cells, represented a very small population (1.9%) and showed a low proliferation activity. Ki-67(+) cells showed a significantly different CK expression pattern compared with that of Ki-67(−) cells. The nuclear areas of CK5(−)/CK20(+) cells (71.3 ± 25.9 μm2) were significantly larger than those of CK5(+)/CK20(+) cells (66.6 ± 25.5 μm2). Negativity for CK5 was related to the grade of UC and an increased number of CK5(−)/CK20(+)/Ki-67(+) cells was related to a higher malignant potential. We conclude the nuclear morphology is related to cell differentiation represented by CK expression and cell proliferative activity.