Abstract Purpose: Angiogenesis and vascular endothelial growth factor (VEGF) expression are associated with a poor outcome in bladder cancer. To understand more about the mechanisms, we studied the role of delta-like 4 (DLL4), an endothelial-specific ligand of the Notch signaling pathway, in bladder cancer angiogenesis. Experimental Design: The expression of DLL4, CD34, and VEGF were studied in a cohort of 60 bladder tumors and 10 normal samples using quantitative PCR. In situ hybridization was used to study the pattern of DLL4 expression in 22 tumor and 9 normal samples. Serial sections were also stained for CD34 and α-smooth muscle actin (α-SMA) using conventional immunohistochemistry. Results: The expression of DLL4 was significantly up-regulated in superficial (P < 0.01) and invasive (P < 0.05) bladder cancers. DLL4 expression significantly correlated with CD34 (P < 0.001) and VEGF (P < 0.001) expression. The in situ hybridization studies showed that DLL4 was highly expressed within bladder tumor vasculature. Additionally, DLL4 expression significantly correlated with vessel maturation as judged by periendothelial cell expression of α-SMA, 98.7% of DLL4-positive tumor vessels coexpressed α-SMA, compared with 64.5% of DLL4-negative tumor vessels (P < 0.001). High DLL4 expression may have prognostic value in superficial and invasive bladder. Conclusion: DLL4 expression is associated with vascular differentiation in bladder cancer; thus, targeting DLL4 may be a novel antiangiogenic therapy.