Antioxidant activity of isorhamnetin 3-O neohesperidoside (I3ON), isolated from the leaves of Acacia salicina, was determined by the ability of this compound to inhibit lipid peroxidation and to protect against hydroxyl radical-induced DNA damage in pKS plasmid DNA and Escherichia coli cultures. Antigenotoxic activity was assessed by using the comet assay. The IC(50) value of the inhibitory activity toward lipid peroxidation by I3ON is 0.6 mM. This compound was also able to protect against hydroxyl radical-induced DNA damage in pKS plasmid DNA. Moreover, this compound induced an inhibitory activity toward H2O2-induced genotoxicity. The protective effect exhibited by this molecule was also determined by analysis of gene expression as a response to an oxidative stress, using a cDNA microarray. Transcription of several genes related to the antioxidant system (HMOX2 and TXNL) and to the DNA repair pathway (XPC, POLD1, POLD2, PCNA, DDIT3, APEX, and LIG4) were upregulated after incubation with I3ON. Taken together, these observations provide evidence that the I3ON, isolated from the leaves of A. salicina, is able to protect cells against oxidative stress.