The aim of this study was to study the expression profiles of muscle-invasive bladder cancer (MIBC) cells of different risk groups and to explore the crucial role of biological pathway change in heterogeneity of MIBC cells. Thirty individual samples (cancer and non-cancerous specimens) were obtained from patients with MIBC. Laser capture microdissection was employed to harvest the homogeneous MIBC cells and normal urothelial cells. iTRAQ and 2D-LC-MS/MS were used to quantify and identify the differently expressed proteins. Then, the significantly changed proteins were further analyzed using Arraytrack ™ software. The interested proteins were compared with the published literatures to discuss the exact functions. A total of 3,073 non-redundant proteins were identified in this research; therefore, 855/2,210/633 (fold change >1.5 relative to normal group) presented in high-/median-/low-risk groups, respectively. 617/1,620/463 proteins with SWISS-ACC number output from Arraytrack ™ software and presented in high-/median-/low-risk groups, respectively. Pathway analysis revealed that the mainly changed pathways (top-10, p < 0.05) in Genetic information processing category were similar in high- and median-risk groups, including Kyoto Encyclopedia of Genes and Genomes (KEGG) spliceosome, protein export, ribosome pathways. The mainly altered pathways in Metabolism category included glycolysis/gluconeogenesis, pentose phosphate, pyruvate metabolism pathway for high-risk group, and glutathione metabolism, citrate cycle, oxidative phosphorylation pathways for median-risk group. The major changed pathways for low-risk group included focal adhesion pathway and ECM-receptor interaction pathway. The changed biological pathways are closely related to the regulation of heterogeneity for MIBC. The KEGG pathways of Genetic information processing category and Metabolism (anaerobic or aerobic) category play a crucial role in determining the malignant phenotype of MIBC cells. The quantification analysis of proteins combining with the KEGG pathway analysis contributes to screening candidate biomarkers and guides the biological molecular therapy of MIBC.