Eupatorium adenophorum Spreng., a major invasive weed in southwestern China, has caused great economic losses. In order to find a new herbicide to control E. adenophorum, experiments were conducted to study its physiological and biochemical responses to low and high doses of picloram herbicide (4-amino-3,5,6-trichloropyridine-2-carboxylic acid). Electrolyte leakage, malondialdehyde, and free proline were stimulated by picloram, showing a remarkably increase (p < 0.05) with high herbicide concentration (60, 120, and 240 g ai ha-1). The treated plants exhibited lower osmotic adjustment capacity, high dosage lipid peroxide levels and more free-proline accumulation. It was found that low doses (12 and 24 g ai ha-1) of picloram initially increased catalase, peroxidase, superoxide dismutases and protein, but these indicators decreased (p < 0.05) with the increase of treating time (after 3 d) and dose (120 and 240 g ai ha-1). In addition, the structures of chloroplasts and mitochondria were seriously deformed. These results indicated E. adenophorum can improve its herbicide-tolerance by increasing the antioxidative system activity at the initial period of low picloram stress. However, this protective function disappeared with increasing of treating time and picloram dosage. Eupatorium adenophorum responded differently to low and high concentrations of picloram and ultrastructural changes are an important cause of death in E. adenophorum.