In the present paper, the influence of a {101¯2}–{101¯2} secondary twin on detwinning of a {101¯2} primary twin is systematically investigated by compression along the normal direction (ND) of pre-strained samples at room temperature. Samples containing both a {101¯2} primary twin and a {101¯2}–{101¯2} 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 {101¯2}–{101¯2} secondary twin into the primary twin can enhance the compression yield strength along the ND. During compression along the ND, the {101¯2}–{101¯2} secondary twin mainly deforms by {101¯2} twinning, forming a {101¯2}–{101¯2}–{101¯2} ternary twin, while detwinning of the {101−2} primary twin also takes place. When a {101¯2}–{101¯2} secondary twin happens to totally separate a {101¯2} primary twin from the matrix, it can serve as a barrier to impede detwinning of this {101¯2} primary twin. The {101¯2} twinning in a {101¯2}–{101¯2} secondary twin can induce a preferred distribution of prismatic planes in twinned regions, which is closely related to the activation of preferential twin variants.