The actions of luteinizing hormone (LH) to induce ovulation and luteinization of preovulatory follicles are mediated principally by activation of cAMP-dependent protein kinase (PKA) in granulosa cells. PKA activity is targeted to specific locations in many cells by A-kinase anchoring proteins (AKAPs). We previously showed that follicle-stimulating hormone (FSH) induces expression of microtubule-associated protein (MAP) 2D, an 80-kDa AKAP, in rat granulosa cells, and that MAP2D co-immunoprecipitates with PKA regulatory subunits in these cells. Here we report a rapid and targeted dephosphorylation of MAP2D at Thr256/Thr259 after treatment with human chorionic gonadotropin (hCG), an LH receptor agonist. This event is mimicked by treatment with forskolin or a cAMP analog and is blocked by the PKA inhibitor myristoylated-PKI, indicating a role for cAMP and PKA signaling in phospho-regulation of granulosa cell MAP2D. Furthermore, we show that Thr256/Thr259 dephosphorylation is blocked by the protein phosphatase 2A (PP2A) inhibitor okadaic acid and demonstrate interactions between MAP2D and PP2A by co-immunoprecipitation and microcystin-agarose pulldown. We also show that MAP2D interacts with glycogen synthase kinase (GSK) 3 and is phosphorylated at Thr256/Thr259 by this kinase in the basal state. Increased phosphorylation of GSK3 at Ser9 and the PP2A B56 subunit at Ser566 is observed after treatment with hCG and appears to result in LH receptor-mediated inhibition of GSK3 and activation of PP2A, respectively. Taken together, these results show that the phosphorylation status of the AKAP MAP2D is acutely regulated by LH receptor-mediated modulation of kinase and phosphatase activities via PKA.