Inhibition of Notch signaling pathways, consisting of 4 highly conserved receptors (Notch 1-4), induces expression of Krüppel-like transcription factors (KLFs) linked to bladder cancer tumorigenesis and metastasis. Effects of Notch-1 knockdown on cell proliferation, differentiation and KLF4 levels in bladder cancer cell lines were investigated. PsiRNA1‑mediated Notch-1 and KLF4 knockdown models and control model without the psiRNA1 vector were constructed using bladder cancer cell lines T24 and BIU87. Cell proliferation, apoptosis and expression of Notch-1 and KLF4 were assessed using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, flow cytometry assay with Annexin V-FITC/PI staining, and reverse transcriptase polymerase chain reaction (RT-PCR) and western blot analysis, respectively. Proliferation was assessed in Notch-1 and/or KLF4 knockdown. The results showed that Notch-1 mRNA and protein expression levels were significantly lower following psiRNA1 vector transfection in both cell lines (P<0.05). Growth and proliferation of both cell lines were significantly inhibited by Notch-1 knockdown (P<0.05), and more G0/G1 arrest and apoptosis were observed compared to those in the control groups (P<0.05). The effects were time-dependent, peaking between 24-48 h and declining by 72 h. KLF4 expression was significantly higher in the Notch-1 knockdown group than in control cells (P<0.05). Notch-1 knockdown cell proliferation was significantly lower than that of Notch-1 and KLF4 knockdown (P<0.05). In conclusion, Notch-1 may act as an oncogene, regulating the proliferation and differentiation of bladder cancer cells by inhibiting KLF4. Pending further exploration of pathway variations and crosstalk, these pathways may be useful targets for bladder cancer therapy.