Vasoactive intestinal peptide (VIP) is a neurotransmitter that primarily functions as a vasodilator. VIP plays its role through binding to its receptors known as VIP/pituitary adenylate cyclase-activating peptide receptors (VPACs). In this study, we examined the expression of VPAC1 in human colon cancer tissues, analyzed the relationship between VPAC1 expression and cancer malignancy, and explored the possible mechanisms using immunohistochemistry and immunofluorescence double staining. The results showed that (1) poorly differentiated colon cancers have significantly higher VPAC1 expression than well-differentiated colon cancers do (p < 0.01); (2) phospho-epithelial growth factor receptor (EGFR) overexpression/activation in the cytoplasm of cancer cells is related to VPAC1 overexpression; (3) blood vessels surrounding colon cancer have significantly more VPAC1-positive than normal colon mucosa does; (4) tumor-associated macrophages (TAMs) of colon cancer have a higher level of VPAC1 expression than macrophages in normal colon mucosa do. These data suggest that VPAC1 overexpression is associated with poorer differentiation of colon cancer, which is likely caused by subsequent EGFR activation in cancer cells. In addition, VPAC1 overexpression in both blood vessels and macrophages in tumors may also play an important role in the development of aggressive cancer.