〔Abstract〕 Objective To investigate whether fullerene C60 administered by intratracheal spraying promotes N-bis (2-hydroxypropyl) nitrosamine (DHPN) induced-lung tumor development and the underlying mechanisms. Methods A rat lung tumor model was induced by the DHPN drinking water method. Eight-week-old male Fisher 344 rats were randomly divided into 5 groups (n = 15): DHPN+Veh group (0.2% DHPN + vehicle solution), DHPN+C60-L group (0.2% DHPN + 250 μ g/mL C60), DHPN+C60-H group (0.2% DHPN + 500 μg/mL C60), DHPN group (0.2% DHPN), and C60-H group (normal drinking water + 500 μg/mL C60). 0.2% DHPN in drinking water was given to initiate carcinogenesis, and 2 weeks later, different concentrations of fullerene C60 were administered by intratracheal spraying every two weeks for a total of 20 times. Two weeks after the last spraying, the incidence, number and size of lung tumors were statistically analyzed. In another mechanism animal experiment, 8-week-old male Fisher rats were divided into two groups with 5 rats in each group: C60 group and the control vehicle group. 500 μg/mL C60 or the control vehicle intratracheally sprayed every two days for a total of 5 times. Six hours after the last spraying, the lungs were removed and used for observation ofC60 distribution and for detection of SOD, 8-OHdG and cytokines in lung tissues. Results No lung tumorigenesis was observed in the C60-H group; the incidence, number and size of lung tumors in the DHPN+C60-H group were significantly higher than those in the DHPN+Veh group (P < 0.05, P < 0.001, P < 0.05) and the DHPN group (P < 0.01, P < 0.001, P < 0.01), respectively. In additional animal experiments designed for mechanistic investigation, the levels of SOD and 8-OHdG in the experimental group were markedly elevated compared with the control group (both P < 0.001), with statistically significant differences. Conclusion Intratracheal nebulization of high-concentration fullerene C60 promoted DHPN- induced lung tumorigenesis; C60 led to an increase in reactive oxygen species (ROS) production, thereby elevating the potential for gene mutation. This may represent one of the mechanisms underlying C60-induced promotion of lung tumorigenesis.