In the present paper, the influence of a – secondary twin on detwinning of a primary twin is systematically investigated by compression along the normal direction (ND) of pre-strained samples at room temperature. Samples containing both a primary twin and a – secondary twin are prepared by pre-compression along the transverse direction (TD) and subsequent re-compression along the rolling direction (RD) of a hot-rolled AZ31 Mg alloy plate at room temperature. Our results show that the introduction of a – secondary twin into the primary twin can enhance the compression yield strength along the ND. During compression along the ND, the – secondary twin mainly deforms by twinning, forming a –– ternary twin, while detwinning of the primary twin also takes place. When a – secondary twin happens to totally separate a primary twin from the matrix, it can serve as a barrier to impede detwinning of this primary twin. The twinning in a – secondary twin can induce a preferred distribution of prismatic planes in twinned regions, which is closely related to the activation of preferential twin variants.