Abstract: Objective To explore the inhibitory effect and mechanism of midazolam on transplanted tumor of nude mice with human lung cancer A549 cells. Methods Totally 12 nude mice model of human lung cancer A549 cells were established and randomly divided into midazolam group(n=6)and control group(n=6). Dosage of 0.8 mg/kg midazolam was injected intraperitoneally once a day in midazolam group, and saline was injected in control group. After 20 days, the body mass and the volume of transplanted tumor were observed. Nude mice were sacrificed, and HE staining was used to observe the effect of midazolam on the cell morphology of transplanted tumor tissue. TUNEL staining was used to detect tumor cell apoptosis in transplanted tumor tissues, immunohistochemical(IHC)staining was used to observe the changes in Ki-67 in tumor tissue cells, and Western-blot detection was used to observe the changes of signal transducer and activator of transcription 3(STAT3)proteins in transplanted tumor tissue. Results Compared with the control group, the tumor mass and volume in midazolam group were significantly smaller(P<0.05 or P<0.01). HE staining showed that the tumor tissue of midazolam group had a large area of necrosis. TUNEL staining showed that the apoptosis number of midazolam group was significantly higher than that of the control group, IHC staining showed that midazolam can significantly reduced the expression of Ki-67 protein in the tumor tissue after intervention, midazolam can significantly reduced the expression of - Ki-67 protein in the tumor tissue, and the expression of STAT3 protein in midazolam group regulated down significantly(P<0.05). Conclusion Midazolam can significantly inhibit the growth of A549 transplanted tumor tissue and induce tumor cell apoptosis by regulating STAT3 expression.