Cucurbitacin IIb (CuIIb) extracted from Hemsleya penxianensis has been demonstrated anticancer activity in many malignancies, however, its effect against bladder cancer cells and the molecular mechanism remains unclear. Accordingly, in the present study, we evaluated the effect and further the underlying mechanism of CuIIb on bladder cancer cells. Cell viability and clonogenicity were examined to evaluate growth suppressive effect of CuIIb, alongside mechanism exploration was conducted based on RNA sequencing (RNA-seq). The results showed that CuIIb exposure inhibited the growth of T24 and UM-UC-3 bladder cancer cells as indicated by its obvious suppression on cell viability and clonogenicity. Mechanistic studies by RNA-seq and quantifying analysis of RNA-seq data by TMNP indicated cell cycle modulated by cell cycle checkpoints and apoptosis mediated by PI3K/Akt pathway might account for the anticancer activity of CuIIb. Consistently, results of flow cytometry and AO/EB staining demonstrated that the growth-suppressive effect of CuIIb was mediated by cell cycle arrest in G2/M phase and robust induction of cell apoptosis, which was further confirmed by immunoblotting and mitochondrial membrane potential ??m) analysis. Collectively, the results presented herein indicated that CuIIb exhibited anticancer activity on bladder cancer which may be a potential candidate for improving bladder cancer outcomes.