The volatile oil from Rhizoma Ligustici Chuanxiong Hort. (CXVO) is likely to be the mainly active ingredient of Chuangxiong in curing headache. In this study, oral administration of CXVO (45.0, 90.0, and 135.0 microl/kg) to mice significantly elevated the pain threshold in the hot-plate test and reduced the number of abdominal writhing caused by acetic acid. CXVO (90.0 and 135.0 microl/kg) not only reduced locomotor activity, but also prolonged the sleeping time induced by sodium pentobarbital (35 mg/kg), and the number of mice with sleeping time over 1 min by sodium pentobarbital (25 mg/kg) was markedly enlarged by CXVO (45.0, 90.0, 135.0 microl/kg) administration. The three doses of CXVO significantly increased the pain threshold of rabbits with headache due to hot radiation and the level of plasma ET of rats with headache due to nitroglycerin injection. Besides, for the nitroglycerin-induced headache rats, the c-fos gene expression in the brain stem and hypothalamus was remarkably inhibited and the level of plasma CGRP was reduced significantly after CXVO administration at both doses 90.0 and 135.0 microg/kg. The latter dosage could also raise the level of plasma 5-HT markedly. The study suggests that CXVO acts probably as the active ingredient of Rhizoma Ligustici Chuanxiong Hort. (CX) on treating headache and has potential to be an agent for treating headache.