The topoisomerase I inhibitor topotecan (TPT) is used in the therapy of different tumors including high-grade gliomas. We previously showed that TPT-induced apoptosis depends on p53 with p53 wild-type (wt) cells being more resistant because of p53-controlled degradation of topoisomerase I. Here, we show that p53-deficient (p53(-/-)) fibroblasts undergo excessive mitochondrial apoptosis featuring H2AX phosphorylation, Bcl-x(L) decline, cytochrome c release, caspase-9/-3/-2 activation, and cleavage of Bid. In wt and apaf-1(-/-) cells, caspase-2 did not become activated and Bid was not cleaved. In addition, p53(-/-) cells cotreated with TPT and caspase-3 inhibitor showed neither caspase-2 activation nor Bid cleavage, implying that caspase-2 is processed downstream of the apoptosome by caspase-3. Although processing of caspase-9/-3 was similar in wt and p53(-/-) cells, only p53(-/-) cells displayed active caspase-3. This was due to the proteasomal degradation of X-chromosome-linked inhibitor of apoptosis (XIAP) and survivin that inhibits caspase-3 activity. Accordingly, TPT-induced apoptosis in wt cells was increased after XIAP/survivin knockdown. Silencing of Bid led to reduction of TPT-triggered apoptosis. Data obtained with mouse fibroblasts could be extended to human glioma cells. In U87MG (p53wt) cells cotreated with TPT and pifithrin-alpha, or transfected with p53-siRNA, caspase-2 and Bid were significantly cleaved and XIAP/survivin was degraded. Furthermore, the knockdown of XIAP and survivin led to increased TPT-triggered apoptosis. Overall, the data show that p53-deficient/depleted cells are hypersensitive to TPT because they down-regulate XIAP and survivin, and thus amplify the intrinsic apoptotic pathway via caspase-3-mediated Bid cleavage. Therefore, in gliomas harboring wild-type p53, TPT-based therapy might be improved by targeted down-regulation of XIAP and survivin.