The Human Embryonic Kidney (HEK) 293 cell line is widely used in research work such as vaccine production, adenovirus and adeno-associated viral vectors, and gene therapy. However, little attention was drawn to the passage level of 293 cells. We first claim that the tumorigenicity of the HEK 293 cell line reached 100% when the passage exceeded 65, whereas using low-passage (<52) HEK 293 cell line no tumor could be induced under the same condition. Results from nude mice assay, tumor tissue histological examination, primary culture, PCR and isoenzyme analysis showed that the tumor in nude mice could only be induced by viable high-passage 293 cells. This suggests that more attention should be paid to the passage level of the HEK 293 cell line, especially for vaccine production but the low-passage HEK 293 cell line should be acceptable to regulatory authorities for recombinant virus vector, vaccines, and gene therapy. Meanwhile, we also find that high-passage HEK 293 can be employed as a highly malignant tumor model as its tumorigenicity increases significantly.