Histamine stimulates uterine contraction, however little is known regarding the mechanism or regulation of uterine histamine receptor signaling. Here we investigated the regulation of G_q/11-coupled histamine receptor signaling in human myometrial smooth muscle cells using the inositol 1,4,5-trisphosphate (IP₃) biosensor eGFP-PH_PLC and the Ca²⁺-sensitive dye Fluo-4. Histamine addition caused concentration-dependent increases in IP₃ and [Ca²⁺]_i in the ULTR human uterine smooth muscle cell line and primary human myometrial cells. These effects were completely inhibited by the H₁ histamine receptor antagonist, diphenhydramine, and were unaffected by the H₂ histamine receptor antagonist, cimetidine. ULTR and primary myometrial cells were transfected with either dominant-negative G protein-coupled receptor kinases (GRKs) or siRNAs targeting specific GRKs to assess the roles of this protein kinase family in H₁ histamine receptor desensitization. Dominant-negative GRK2, but not GRK5 or GRK6, prevented H₁ histamine receptor desensitization. Similarly, transfection with siRNAs (that each caused > 70% depletion of the targeted GRK) for GRK2, but not GRK3 or GRK6, also prevented H₁ histamine receptor desensitization. Our data suggest that histamine stimulates phospholipase C-signaling in myometrial smooth muscle cells through H₁ histamine receptors and that GRK2 recruitment is a key mechanism in the regulation of H₁ histamine receptor signaling in human uterine smooth muscle. These data provide insights into the in situ regulation of this receptor subtype and may inform pathophysiological functioning in pre-term labor and other conditions involving uterine smooth muscle dysregulation.