Non-small cell lung cancer (NSCLC) is a serious threat to people's health. This study aims to determine the possible effect of Gujin Xiaoliu Tang (GJXLT) on NSCLC, which is an empirical formula from Professor Dai-Han Zhou. In this study, chromatographic fingerprinting of GJXLT and A549 cell model in vitro and in vivo was established. We cultured A549 cells in vitro and found that GJXLT inhibited A549 cell growth and induced apoptosis. Compared with the control group, the expression of p-STAT3 and VEGF proteins in the GJXLT groups was decreased. Similar findings were also observed in vivo. First, GJXLT inhibited the growth of transplanted tumor and did not reduce the weight of the tumor-bearing mice in comparison with that of the control group. Then, the Ki-67 expression of transplanted tumor in the GJXLT groups was decreased. In addition, the apoptosis rate of transplanted tumor in the GJXLT groups was increased. Overall, our data showed that GJXLT inhibited A549 cell proliferation and induced apoptosis in vivo and in vitro. Furthermore, GJXLT inhibited the growth of lung cancer xenograft in nude mice model with no obvious side effects. The anti-tumor effect of GJXLT might also be related to the inhibition of p-STATS and VEGF expression in the JAK2/STAT3 pathway. Our results demonstrated the potential of GJXLT as a novel treatment for NSCLC.