β-cembrenediol ((1S,2E,4R,6R,7E,11E)-2,7,11-cembratriene-4,6-diol), shown to be one of the most important allelochemicals of tobacco in previous studies, effectively inhibited the root and stem growth of receptor plants and the inhibitory effects was concentration-dependent. However, its mechanism of action remains unclear. Seedlings of lettuce (Tai Yuan Sun) were treated withβ-cembrenediol to clarify its mode of action. Results showed that β-cembrenediol signifcantly inhibited the seedling growth and reduced fresh weight of L. sativa. The compounds effectively affected cell mitotic index and caused cell death. Exposure to β-cembrenediol induced overproduction of reactive oxygen species (ROS). Moreover, increased content of hydrogen peroxide, malondialdehyde, and proline, and decreased in chlorophyll content indicated lipid peroxidation and induction of oxidative stress. These results suggested thatβ-cembrenediol caused oxidative damage through enhanced generation of ROS, as indicated by increased lipid peroxidation, disruption of membrane integrity and impacted mitosis, ultimately resulted in growth inhibition of the receptor plant.