Update publication information. Morphogenesis of many protozoans depends on a polarized establishment of cortical cytoskeleton possessing the subpellicular microtubules (SPMTs), which are apically nucleated and anchored by the apical polar ring (APR). In the malaria parasite Plasmodium, APR can be observed when a round zygote differentiates to a crescent motile ookinete for mosquito midgut infection. So far, the fine structure and molecular components of APR as well as the underlying mechanism of APR-mediated apical positioning of SPMTs are largely unknown. Here, we report that a previously undescribed MT-binding protein APR2 localizes at APR and associates directly with apical SPMTs throughout ookinete morphogenesis. We resolve an unprecedented APR structure composed of a top ring plus approximate 60 radiating spines, with each spine fitting with an individual SPMT. APR2 disruption impairs ookinete morphological development and gliding motility, leading to failure of mosquito transmission of Plasmodium. The APR2-deficient ookinetes display defective apical anchorage of APR and SPMT due to the impaired integrity of APR. Using proximity labeling with APR2 as bait, we obtain a Plasmodium ookinete APR proteome for the first time and validate 10 new APR proteins. Among the proteins, APRp2 and APRp4, directly interacting with APR2, also mediate the apical anchorage of SPMTs. This study sheds light on the molecular basis of APR in the organization of SPMTs essential for ookinete morphogenesis and mosquito transmission of Plasmodium.