Abstract: Gynostemma pentaphyllum（GYP） is a traditional Chinese medicinal material which has a significant effect on a variety of cancers. To screen the target of GYP on bladder cancer, this study used 30 SPF grade SD rats to construct model group, drug group and blank group, and then establish bladder cancer model by injecting N-methyl-N-nitrosourea（MNU） and used the same way to treat by GYP. All of the group were taken the tissue at the10 thweek, and the bladder tumors in each group were observed by H＆E staining pathological sections. Proteomics tandem mass spectrometry（TMT） 10-fold labeling method was used to detect differentially expressed proteins in the bladder tissue of each group, and the targets were screened in combination with the three groups of bladder cancer data sets in the GEO database. The results showed that GYP reversely regulated 335 proteins, including 55 up-regulated and 280 down-regulated. GEO’s three differentially expressed proteins（DEGs） pooled in three bladder cancer expression profiles include 20 up-regulations and 50 down-regulations. By screening DEGs in GEO and proteins with the same bladder cancer trend in TMT proteomics, combined with GYP reverse regulation data, a total of three reverse regulation targets were obtained, including one down-regulated target CNN1 and two up-regulated targets KRT19 and PCP4, and then checked by Western blot. The results of the study indicate that CNN1,KRT19 and PCP4 may be potential targets for the treatment of bladder cancer by GYP, and GYP may resist bladder cancer by regulating CNN1, KRT19 and PCP4, which provided molecular basis for the treatment of bladder cancer by GYP.