Bladder cancer is a common urologic cancer whose incidence continues to rise annually. Urinary microparticles are an attractive material for noninvasive bladder cancer biomarker discovery. In this study, we applied isotopic dimethylation labeling coupled with liquid chromatography-tandem mass spectrometry (LC-MS/MS) to discover bladder cancer biomarkers in urinary microparticles isolated from hernia (control) and bladder cancer patients. This approach identified 2964 proteins based on more than two distinct peptides, of which 2058 had not previously been reported as constituents of human urine exosomes/microparticles. A total of 107 differentially expressed proteins were identified as candidate biomarkers. Differences in the concentrations of 29 proteins (41 signature peptides) were precisely quantified by LC-MRM/MS in 48 urine samples of bladder cancer, hernia, and urinary tract infection/hematuria. Concentrations of 24 proteins changed significantly (p<0.05) between bladder cancer (n=28) and hernia (n=12), with area-under-the-curve values ranging from 0.702 to 0.896. Finally, we quantified tumor-associated calcium-signal transducer 2 (TACSTD2) in raw urine specimens (n=221) using a commercial ELISA and confirmed its potential value for diagnosis of bladder cancer. Our study reveals a strong association of TACSTD2 with bladder cancer and highlights the potential of human urinary microparticles in the noninvasive diagnosis of bladder cancer.