Gold nano rods (GNRs) have showed cytotoxicity to cancer cells. At the same time, it shows little effects on non-tumor cells. Between GNRs and sub-cellular organelles, the understanding of interaction plays a very important role to determine the intracellular mechanisms. The purpose of what we done is to explain the effects of the surface properties of GNRs on specific cancer cell death. Three GNR samples with different aspect ratios were finely prepared by the seed-mediated growth method. Then the intracellular transport and the in vitro/vivo mechanisms of cancer cell death were studied by transmission electron microscopy (TEM), confocal laser scanning microscopy (CLSM), laser light scattering, and flow cytometry (FCM). It was found that GNRs700 exhibited the largest photothermal conversion efficiency. However, the GNR660 with or without light stimulation exhibited the highest cytotoxicity against cancer cells, which was contradict to the general knowledge. Detailed intracellular investigations showed that the lysosome was the key sub-organelle affecting the GNR function. Further experiments revealed that cytotoxicity was strongly affected by the GNR's surface potential. This potential was actually related to the density of surface cationic molecules, which further regulated lysosomal membrane penetration. The results obtained herein indicated that the physicochemical properties of the surface potential mediated the specific toxicity of GNRs against tumours.