Bladder cancer is the fifth most common malignancy in the world and represents the second most common cause of death among genitourinary tumors. Current prognostic parameters such as grade and stage cannot predict with certainty the long-term outcome of bladder cancer and as a result, there is a pressing need to identify markers that may predict tumor behavior. Earlier we identified the adipocyte fatty acid binding protein (A-FABP), a small molecular-weight fatty acid-binding protein that functions by facilitating the intracellular diffusion of fatty acids between cellular compartments, as a putative marker of progression based on a limited study of fresh bladder urothelial carcinomas (UCs). Here we have comprehensively examined the protein expression profiles of a much larger sample set consisting of 153 bladder specimens (46 non-malignant biopsies, 11 pTa G1, 40 pTa G2, 10 pTa G3, 13 pT1 G3, 23 pT2-4 G3, and 10 pT2-4 G4) by gel-based proteomics in combination with immunohistochemistry (IHC) using a peptide-based rabbit polyclonal antibody that reacts specifically with this protein. Proteomic profiling showed a striking down-regulation of A-FABP in invasive lesions, a fact that correlated well with immunohistochemical analysis of the same samples. The IHC results were confirmed by using a tissue microarray (TMA) containing 2317 samples derived from 1849 bladder cancer patients. Moreover, we found that the altered expression of A-FABP in invasive UCs is not due to deregulated expression of peroxisome proliferator-activated receptor (PPAR), a trans-activator of A-FABP. Taken together, these results provide evidence that deregulation of A-FABP may play a role in bladder cancer progression, and suggest that A-FABP could have a significant prognostic value in combination with other biomarkers.