In this study, we show that interfering with the splicing machinery results in activation of the tumour-suppressor p53. The spliceosome was targeted by small interfering RNA-mediated knockdown of proteins associated with different small nuclear ribonucleoprotein complexes and by using the small-molecule splicing modulator TG003. These interventions cause: the accumulation of p53, an increase in p53 transcriptional activity and can result in p53-dependent G(1) cell cycle arrest. Mdm2 and MdmX are two key repressors of p53. We show that a decrease in MdmX protein level contributes to p53 activation in response to targeting the spliceosome. Interfering with the spliceosome also causes an increase in the rate of degradation of Mdm2. Alterations in splicing are linked with tumour development. There are frequently global changes in splicing in cancer. Our study suggests that p53 activation could participate in protection against potential tumour-promoting defects in the spliceosome. A number of known p53-activating agents affect the splicing machinery and this could contribute to their ability to upregulate p53. Preclinical studies indicate that tumours can be more sensitive than normal cells to small-molecule spliceosome inhibitors. Activation of p53 could influence the selective anti-tumour activity of this therapeutic approach.