Abstract: Based on the L₁₆(4⁴) orthogonal test of shoot cutting propagation, the most optimized multi-factors combination for the propagation can be recognized. Through anatomical and physiological studies, the changes of anatomical structure and physiological response mechanism of shoot rooting of Lavandula angustifolia were revealed. The results show that the most optimal level combination for shoot cutting propagation of L. angustifolia is A₃B₁C₂D₃(the ratio of the proportion of substrate pearlite and peat soil is 6∶4 and cutting taken from the top, cutting length was 10 cm, ABT concentration to 1000 mg/kg, with 100% root balls forming and 19 d spending). The cuttings position on branch make great effects for root balls forming rate. Root primordia is originated by induced pattern, and mixed rooting mode produce the roots. The callus formations of the cutting base are independent from the adventitious roots production. Soluble protein, MDA and SOD were significantly correlated or very significantly correlated, and POD was significantly correlated with SOD and MDA. The rooting effect of plant cutting propagation is not necessarily related to the existence of latent root primordium. The metabolism of nutrition responding to plant enzyme protection system activity and MDA under various environmental stresses during the different development stages of root cutting is the predominant factor for rooting.