Ca2+-permeable transient receptor potential canonical 3 (TRPC3) channel is involved in memory modulation by regulating the excitability of hippocampal neurons, contributing to enhanced afterhyperpolarization (AHP). Calcium-activated potassium channels are fundamental modulators of neuronal excitability and contribute to the generation of AHPs following action potential bursts. TRPC3 channels enhance the activity of Ca2+-dependent K+ channels by local, microdomain communication.To uncover downstream targets of TRPC3 signaling in hippocampal neurons, we performed electrophysiological recordings, fluorescent imaging, and SiMPull.TRPC3 channel stimulation with a selective TRPC3/6/7 agonist, GSK1703924A (GSK) led to a reversible decrease in the firing frequency of cultured neurons.Conversely, GSK did not exhibit any effects on the excitability of hippocampal neurons derived from TRPC1-7 knockout mice. Ca2+ levels in Fluo-4 loaded dissociated hippocampal neurons revealed an increase upon application of GSK. Subsequently, we investigated the relationship between these channels using transiently transfected HEK293 cells.Calcium imaging conducted on HEK293 cells overexpressing TRPC3, large-conductance (BKCa), and small-conductance (SKCa) KCa unveiled a notable impairment in cytoplasmic calcium loading specifically in cells co-expressing TRPC3 and SKCa. Furthermore, co-expression of TRPC3 and SKCa induced significant membrane hyperpolarization compared to TRPC3 alone in the presence of extracellular calcium. Conversely, exclusion of calcium from the buffer or employing a Ca2+-impermeable mutant TRPC3 (E630Q) depolarized plasma membrane in transfected HEK293 cells. Moreover, SiMPull demonstrated a physical TRPC3 and SKCa channel interaction.Both constitutive and transient TRPC3 activity serve as a calcium source that specifically enhances SKCa currents, rather than BKCa currents, to regulate excitability of hippocampal neurons.