The concentration and composition of plant secondary compounds are well known to confer plant resistance to herbivores, pathogens and mechanical wounding. Glucosinolates (GSs) are a major group of nitrogen- and sulfur-containing secondary metabolites of Arabidopsis thaliana. The effect of mechanical wounding on GS profile in A. thaliana rosette leaves was reported in this study. A. thaliana ecotype Columbia (Col-0) was grown in a growth chamber. At the twenty-leaf stage, the rosette leaves were wounded by cutting approximately 40% leaf area across the apical lamina with scissors. The leaves were harvested at 0.25, 0.5, 1, 15, 3, 6, 9 and 24 h after the treatment. GSs were extracted from rosette leaves. The dominant GSs were analyzed by HPLC. Wounding by scissors tend to increase GS contents. At 3, 6 and 9 h after treatment, total GS content increased significantly. The contents of both aliphatic GSs and indole GSs were induced at 3h after wounding, e.g., the aliphatic GS levels were increased up to 3.53 fold. The content of 4-methylsulphinylbutyl GS (4MSOB) in rosettes was the highest among all the 12 GSs detected, accounting for 48.5% of total content of GSs. The increase in the content of 4MSOB after wounding is probably the main factor affecting the GS profile in rosette leaves of A. thaliana. Mechanical wounding also changed the proportion of GSs in rosette leaves. The proportion of aliphatic GSs was significantly increased from ca. 66% to 71%-75% at 1, 3, 6, and 9 h after treatment. The proportions of 4MSOB and other GSs displayed significant changes after wounding. Collectively, our results indicate that mechanical wounding regulates glucosinolate profiles in A.thaliana leaves. Further studies of the molecular mechanisms underlying the regulation will lead to advanced understanding of plant defence and interaction with the environment.