Apoptosis has an essential role in the development and homeostasis of all multicellular organisms. In the mitochondrial pathway, cytochrome c is released and together with Apaf-1 leads to the caspase-9 activation. Caspase-9 is responsible for caspase-3 activation. Cleavage of intracellular substrates by caspase-3 results in apoptosis. Smac/DIABLO protein is released from mitochondria as well and is responsible for inhibit the inhibitor apoptosis proteins (IAPs) activity, promoting the apoptosis. Excessive expression of IAPs occurs in several types of cancer cells, what leads to apoptosis resistance in therapies. BIR3 domain of XIAP is responsible for inhibit the activation of caspases-9. The tetrapeptide AVPI from Smac/DIABLO is able to interact with BIR3 domain and increases the sensitivity of cancer cells to chemotherapic agents. Our aim in this study was investigate the stability of the BIR3 domain and its interaction with the peptide AVPI. To promote disturbances in the domain structure, we have utilized high hydrostatic pressure, high temperature and chemical denaturing agents. Conformational changes were monitored by using fluorescence and circular dichroism, and thermodynamic properties were obtained by using isothermal titration calorimetry (ITC). Our results showed that BIR3 is very stable to all treatments. In addition, the tetrapeptide AVPI was able to stabilize the BIR3 domain. The BIR3 domain and the complex BIR3-AVPI were stable until 50°C. Our data of ITC indicate that the interaction occurs in equimolar proportion and that the reaction is exothermic with prevalence of hydrophobic interactions. The stability characterization and the thermodynamic parameters can provide important information for the elucidation of the Smac/DIABLO-IAP interaction and it is consequently important on the development of new drugs that block IAPs, leading cancer cells to apoptosis. Support: CAPES, CNPq, FAPERJ, FUJB/UFRJ e PRONEX.