The effect of nanoparticles on co-cultured tumor and endothelial cells has not been consistently elucidated. To address this question, a microfluidic chip for the co-culture of tumor and endothelial cells is fabricated herein. Human umbilical vein endothelial cells (HUVECs) and human cervical cancer cells (Hela) are respectively cultured in the upper and lower chambers of the chip, and the culture medium with carboxylated nanodiamonds (NDs) is applied to HUVECs by perfusion. The results show that, compared to the individually cultured HUVECs, the HUVECs co-cultured with Hela exhibit cancerous characteristics, with increased leakiness, migration, and proliferation. It is also found that, while the presence of NDs can lead to increased endothelial permeability, the migration and proliferation of the co-cultured HUVECs are reduced, and the co-cultured HUVECs are hindered, to some extent, from becoming cancerous. The results of gene analysis also prove that NDs can inhibit the proliferation of HUVECs by significantly down-regulating the vascular endothelial growth factor (VEGF) gene expression. The mechanism of suppressing tumor growth by inhibiting tumor vascular proliferation was also demonstrated in-vivo using a tumor-bearing mouse model.