<p>cGMP signal-activated ookinete gliding is essential for mosquito midgut infection of <i>Plasmodium</i> in malaria transmission. During ookinete development, cGMP synthesizer GCβ polarizes to a unique localization “ookinete extrados site” (OES) until ookinete maturation and activates cGMP signaling for initiating parasite motility. However, the mechanism underlying GCβ translocation from cytosol to OES remains elusive. Here, we use protein proximity labeling to search the GCβ-interacting proteins in ookinetes of the rodent malaria parasite <i>P. yoelii</i>, and find the top hit Sir2A, a NAD⁺-dependent sirtuin family deacetylase. Sir2A interacts with GCβ throughout ookinete development. In mature ookinetes, Sir2A co-localizes with GCβ at OES in a mutually dependent manner. Parasites lacking Sir2A lose GCβ localization at OES, ookinete gliding, and mosquito infection, phenocopying GCβ deficiency. GCβ is acetylated at gametocytes but is deacetylated by Sir2A for OES localization at mature ookinetes. We further demonstrate that the level of NAD⁺, an essential co-substrate for sirtuin, increases during the ookinete development. NAD⁺ at its maximal level in mature ookinetes promotes Sir2A-catalyzed GCβ deacetylation, ensuring GCβ localization at OES. This study highlights the spatiotemporal coordination of cytosolic NAD⁺ level and NAD⁺-dependent Sir2A in regulating GCβ deacetylation and dynamic localization for <i>Plasmodium</i> ookinete gliding.</p>