p53 has attracted tremendous attention due to its master role in tumor development. Activation of p53 in tumor cells has been the prime focus for cancer drug discovery. Recent studies have shown that few miRNAs can regulate p53 activity directly or indirectly. We herein demonstrate that miR-128 positively regulates p53 activity. Our data suggest that miR-128 inhibits SIRT1 expression directly through a miR-128-binding site within the 3' UTR of SIRT1. miR-128 inhibition of SIRT1 led to an increase in acetylated p53 and its transcriptional targets. miR-128 decreased phospho-Akt and phospho-FOXO3A, increased acetylated FOXO3A and promoted FOXO3A translocation to the nucleus. We further demonstrated that miR-128 augments the antitumor effect of compounds that target the p53 pathway. Furthermore, miR-128 induces apoptosis in wild (WT) p53 as well as in mutant p53-expressing cells in a p53-dependent and -independent manner via induction of PUMA. Pretreatment with PUMA and Bak siRNAs abolished miR-128-induced apoptosis in HCT116 p53+/+ and HCT116 p53-/- cells. Taken together, we present the first evidence of miR-128 to be a new component joining the p53 network. This study emphasizes that miR-128 is a novel mitochondria-targeted miRNA that can be further evaluated as a chemotherapeutic agent for human cancers as it induces apoptosis irrespective of p53 status.