Background and Purpose Tricyclic antidepressants (TCAs) exhibit clinically relevant polypharmacology and interact with multiple molecular targets, including voltage‑gated calcium channels. Although inhibition of CaV3 and CaV2 channels has been reported, the molecular determinants underlying these interactions remain unclear. This study examined how TCAs interact with CaV3 channels relative to CaV2 channels at the channel‑family level. Experimental Approach Automated planar patch‑clamp electrophysiology was used to assess inhibition by amitriptyline, desipramine, nortriptyline, and trimipramine across multiple CaV3 and CaV2 channel isoforms under standardised recording conditions. Amitriptyline and trimipramine were selected for detailed mechanistic analysis at CaV3.3 and CaV2.2 channels. Molecular docking and molecular dynamics simulations were combined with targeted alanine mutagenesis to identify and functionally evaluate pore‑adjacent features associated with TCA sensitivity. Key Results TCAs inhibited both CaV3 and CaV2 channels within the micromolar concentration range, with lower apparent inhibitory thresholds for CaV3 channels. Structural modelling identified pore‑adjacent regions near a conserved CaV3 cysteine (C393 in CaV3.3) where ligand proximity differed between compounds during simulations. No comparable features were identified at the corresponding region of CaV2 channels. Alanine substitution produced compound‑dependent effects, with amitriptyline showing greater sensitivity to C393 substitution than trimipramine. Mutant‑specific analyses also revealed greater variability in substitution effects for amitriptyline. Conclusions and Implications These findings identify channel‑family‑specific structural features associated with differential TCA engagement of CaV3 channels, whereas CaV2 inhibition appears consistent with more distributed cavity interactions. Collectively, the results refine understanding of tricyclic polypharmacology by placing calcium channel modulation within a mechanistic framework.