Phosphatidylinositol and its derivatives are essential components of cell membranes and play pivotal roles in growth signaling pathways. In the human primordial placenta, phosphatidylinositol synthesis is catalyzed by phosphatidylinositol synthase (PIS) and the phosphatidylinositol-exchange enzyme (IE), both of which require divalent cations. We investigated whether GTP-binding proteins modulate this biosynthetic process. Incorporation of [³H]inositol into phosphatidylinositol was measured in trophoblast tissue and microsomes from 8 to 10-week placentas. Our results demonstrate that Mn²⁺ strongly enhances phosphatidylinositol synthesis, and stimulation with AlF₄⁻ further increases incorporation rates by up to 2.5-fold. In contrast, Mg²⁺ combined with the non-hydrolyzable GTP analog GIDP elevated synthesis by 58%, whereas Mn²⁺ plus GIDP reduced incorporation by 30%. Complementary in silico protein–protein interaction analyses suggest that G-proteins may directly associate with inositol-exchange enzymes, providing a potential mechanism for the observed regulatory effects. These findings indicate that phosphatidylinositol synthesis is modulated in a manner consistent with G-protein involvement, with distinct effects depending on the prevailing enzymatic pathway. We propose that rapid trophoblast proliferation may involve feedback mechanisms mediated by distinct G-protein subtypes acting on early steps of the phosphatidylinositol cycle.