Heterosis, characterized by enhanced resistance and yield, has been widely utilized in watermelon breeding. However, our understanding of the regulatory mechanisms underlying male-sterile phenotypes in watermelon remains limited. Here, we determined that the miR159a targets ClMYB33 to regulate anther dehiscence, leading to male sterility in watermelon. Both overexpression of Cl-miR159a (OE-miR159a) and knockout of ClMYB33 (clmyb33) in watermelon suppressed the degradation of septum and stomium t