Tumor vessels often lack the smooth muscle layer, and the instability is conducive to tumor invasion and metastasis. The effect of tumor microenvironment on vascular smooth muscle cells needs to be explored. In the present study, we examined the density of the tumor vessels in human colorectal cancer tissues, and used the tumor conditioned medium of human colorectal cancer HT29 cells to mimic the tumor microenvironment. We showed that the vessel density in colorectal cancer tissues increased, which displayed a decreased expression of smooth muscle a-actin, a specific marker of vascular smooth muscle cells and an attenuated or a discontinuous layer of vascular smooth muscle cells compared with the matched normal tissues. We also showed that the tumor conditioned medium decreased the cell viability, and induced the apoptosis in vascular smooth muscle cells in a concentration-dependent manner. The expression of pro-Caspase-3 was down-regulated, accompanied by increasing of cleaved-Caspase-3 in the cells treated with the tumor conditioned medium, suggesting that Caspase-3 was activated. Moreover, the expression of Bax was increased, and the ratio of Bcl-2/Bax was decreased under the same conditions. Furthermore, the treatment with the tumor conditioned medium resulted in loss of mitochondrial membrane potential in vascular smooth muscle cells. These findings suggest that HT29 cells induce apoptosis of vascular smooth muscle cells in an exocrine manner, associated with activating caspase-3 via mitochondrial apoptotic pathway. This may be one of the mechanisms underlying tumor vascular structural abnormalities.