It has been reported that Salvador (SAV) is a core component of the Salvador-Warts-Hippo (SWH) pathway that restricts cell number, by functioning as a dual regulator of cell proliferation and apoptosis in Drosophila. However, the function of its human ortholog hSav1 (also called hWW45) in mammalian cells is poorly understood. In this study, we identified hematopoietic cell-specific protein 1 (HS1)-associated protein X-1 (HAX1), a 35-kDa protein localized to cell mitochondria, as a novel binding partner of hSav1 using a yeast two-hybrid screening technique. Our finding was confirmed by immunoprecipitation and glutathione-S-transferase (GST) pull-down assays of both proteins. Using immunofluorescence staining, we showed that HAX1 and hSav1 interact with each other. Analysis of the anti-apoptotic function of HAX1 revealed that the presence of hSav1 attenuated the HAX1 protective effects from hydrogen peroxide (H2O2)-induced cell death in MCF-7 cells, while knockdown of hSav1 by small interfering RNAs (siRNAs) significantly enhanced the anti-apoptotic function of HAX1. Also, using the Oncomine database, we found several studies in which HAX1 levels were significantly up-regulated and hSav1 expression was down-regulated in breast cancer samples compared to normal breast tissue. In summary, we conclude that hSav1 interacts with HAX1 and attenuates its protective role against apoptosis in MCF-7 breast cancer cells.