Benzo(a)pyrene (B(a)P), a member of the polycyclic aromatic hydrocarbon family is present ubiquitously in the environment. One of its toxic effects is induction of oxidative stress (mediated by the enzyme B(a)P hydroxylase) which leads to various diseases like cancer. Olive oil (OO) that consists of many antioxidant compounds is reported to have many beneficial properties including protection against cancer. The objective of the present study is to evaluate the effect of OO on B(a)P hydroxylase enzyme and further elucidate the antioxidant capacity of OO against B(a)P-induced toxicity. Rat liver microsomes were divided into three groups: vehicle control, B(a)P treated group, and OO + B(a)P coincubated group. Antioxidant enzymes which were decreased and protein carbonyl content and lipid peroxidation products which were increased on exposure to B(a)P was attenuated to near normal on OO exposure. B(a)P hydroxylase enzyme was very low in OO incubated group which may be due to inhibition of the enzyme by OO or high utilization for the metabolism of B(a)P. Further, no B(a)P metabolites (3-OH B(a)P and B(a)P 7,8-dihydrodiol) were identified in HPLC during B(a)P + OO exposure. The results prove the protective role of OO against B(a)P-induced oxidative damage.