DJ-1 and HSP90α play a significant role in the progression of various types of cancer and are known to be associated with phosphatase and tensin homolog deleted on chromosome 10 (PTEN), PI3K-p110α and pAkt, the signaling molecule proteins from the phosphatidylinositol 3-kinase (PI3K) pathway. However, the expression of these proteins and their clinical significance are not well characterized in urothelial carcinoma (UC). Immunohistochemical analysis of DJ-1, HSP90α, PTEN, pAkt and PI3K-p110α expression was performed on tumor samples from 102 patients with UC to assess the relationship between the expression of each protein and the pathological parameters. The expression of DJ-1 and HSP90α was positively correlated with the pathological stage of UC, whereas PTEN expression negatively correlated with, not only the pathological stage, but also the growth pattern and histological grade of UC. Although PI3K-p110α expression was significantly correlated with DJ-1 as well as PTEN expression in UC, PI3K-p110α expression itself failed to reveal any significant correlation with the clinicopathological parameters. In conclusion, the overexpression of DJ-1 and HSP90α, and a loss of PTEN are associated with invasive UC, and PI3K-p110α expression is correlated with DJ-1 and PTEN expression in UC.