Endothelial-like differentiation of dendritic cells (DCs) is a new phenomenon, and the mechanism is still elusive. Here, we show that the tumor microenvironment derived from the human esophageal squamous cell carcinoma (ESCC) cell line EC9706 can induce immature DCs (iDCs) differentiate toward endothelial cells, and become endothelial-like cells, but it has no obvious influence on mature DCs. During the course of endothelial-like differentiation of iDCs, a sustained activation of mitogen-activated protein kinase/extracelluar signal-regulated kinase1/2 (MAPK/ERK1/2) and cAMP response element-binding protein (CREB) was detected. Incubation of iDCs with MEK phosphorylation inhibitor PD98059 blocked the MAPK/ERK1/2 and CREB phosphorylation as well as the endothelial-like differentiation of iDCs. Inhibition of vascular endothelial growth factor-A (VEGF-A) in the microenvironment with its antibody blocked the endothelial-like differentiation and the phosphorylation of MAPK/ERK1/2 and CREB. These data suggest that MAPK/ERK1/2 signaling pathway activated by VEGF-A could mediate endothelial-like differentiation of iDCs in the ESCC microenvironment.