DJ-1 is a highly conserved protein that protects neurons against oxidative stress and whose loss of function mutations are linked to recessively inherited Parkinson's disease (PD). While a number of signaling pathways have been shown to be regulated by DJ-1, its role in controlling cell survival through non-coding RNAs remains poorly understood. Here, using a microarray screen, we found that knocking down DJ-1 in human neuroblastoma cells results in down-regulation of microRNA-221 (miR-221). This is one of the most abundant miRNAs in the human brain and promotes neurite outgrowth and neuronal differentiation. Yet the molecular mechanism linking miR-221 to genetic forms of PD has not been studied. Consistent with the microarray data, miR-221 expression is also decreased in DJ-1 mouse brains. Re-introduction of wild-type DJ-1, but not its PD-linked pathogenic M26I mutant, restores miR-221 expression. Notably, over-expression of miR-221 is protective against 1-methyl-4-phenylpyridinium (MPP)-induced cell death, while inhibition of endogenous miR-221 sensitizes cells to this toxin. Additionally, miR-221 down-regulates the expression of several pro-apoptotic proteins at basal conditions and prevents oxidative stress-induced up-regulation of bcl-2-like protein 11 (BIM). Accordingly, miR-221 protects differentiated DJ-1 knock-down ReNcell VM human dopaminergic neuronal cells from MPP-induced neurite retraction and cell death. DJ-1 is a known activator of the mitogen-activated protein kinase (MAPK)/extracellular-regulated kinase (ERK) pathway and may modulate miR-221 levels in part through this pathway. We found that inhibiting ERK1/2 decreases miR-221 levels, whereas over-expressing ERK1 in DJ-1 knock-down cells increases miR-221 levels. These findings point to a new cytoprotective mechanism by which DJ-1 may increase miR-221 expression through the MAPK/ERK pathway, subsequently leading to repression of apoptotic molecules. The inability of a pathogenic DJ-1 mutant to modulate miR-221 further supports the relevance of this mechanism in neuronal health and its failure in DJ-1-linked PD.