The NAD+-dependent deacetylase SIRT1 is involved in diverse cellular processes, and has also been linked with multiple disease states. Among these, SIRT1 expression negatively correlates with cancer survival in both laboratory and clinical studies. Active regulator of SIRT1 (AROS) was the first reported post-transcriptional regulator of SIRT1 activity, enhancing SIRT1-mediated deacetylation and downregulation of the SIRT1 target p53. However, little is known regarding the role of AROS in regulation of SIRT1 during disease. Here, we report the cellular and molecular effects of RNAi-mediated AROS suppression, comparing this with the role of SIRT1 in a panel of human cell lines of both cancerous and non-cancerous origins. Unexpectedly, AROS is found to vary in its modulation of p53 acetylation according to cell context. AROS suppresses p53 acetylation only following the application of cell damaging stress, whereas SIRT1 suppresses p53 under all conditions analysed. This supplements the original characterization of AROS but indicates that SIRT1 activity can persist following suppression of AROS. We also demonstrate that knockdown of AROS induces apoptosis in three cancer cell lines, independent of p53 activation. Importantly, AROS is not required for the viability of three non-cancer cell lines indicating a putative role for AROS in specifically promoting cancer cell survival.