To observe the effect of hepatoma carcinoma cells (HepG2) conditioned medium on the morphology and ultrastructure of human umbilical vein endothelial cells (HUVEC). The co-culture system of HepG2 conditioned medium (HepG2-CM) and HUVEC was established. The changes of cell morphology were observed by inverted microscope, and the changes of cell ultrastructure were observed by scanning electron microscopy (SEM), then the roughness of cell membrane surface were analyzed by atomic force microscopy (AFM). The cells in control group depended closely on each other, the edges of the cell membrane were flat, and the cell adhesion arrangement was dense. The connections between the cells in HepG2-CM group were evacuated, the edges of the cell membrane were blurred, and cell adhesion was weakened. The peak to valley roughness (Rp-v) of cell membrane surface of the two groups were (454.83±29.99) nm and (501.28±11.78) nm, P=0.028. Root-mean-square roughness (Rq) were (32.84±4.70) nm and (45.03±3.84) nm, P=0.007. Average roughness (Ra) were (25.06±4.09) nm and (34.39±3.58) nm, P=0.014. Meant Ht were (204.03±11.65) nm and (301.48±12.80) nm, P=0. Median Ht were (206.55±7.46) nm and (313.25±5.89) nm, P=0. The results indicated that HepG2-CM induces the weakening of HUVEC cell adhesion, the evacuation of connections between cells, the formation of “pseudopod-like” protrusions, and the increase of roughness of cell membrane surface, which is helpful for us to further understand the biological behavior of vascular endothelial cells in tumor microenvironment and provide ideas for anti-tumor angiogenesis.