ω-Conotoxins are routinely used as selective inhibitors of different classes of voltage-gated calcium channels (VGCCs) in excitable cells. In the present study, we examined the potent N-type VGCC antagonist ω-conotoxin CVID and non-selective N- and P/Q-type antagonist CVIB for their ability to block native VGCCs in rat dorsal root ganglion (DRG) neurons and recombinant VGCCs expressed in Xenopus oocytes. ω-Conotoxins CVID and CVIB inhibited depolarization-activated whole-cell VGCC currents in DRG neurons with pIC50 values of 8.12 ± 0.05 and 7.64 ± 0.08, respectively. Inhibition of Ba2+ currents in DRG neurons by CVID (∼ 66% of total) appeared to be irreversible for > 30 min washout, whereas Ba2+ currents exhibited rapid recovery from block by CVIB (≥ 80% within 3 min). The recoverable component of the Ba2+ current inhibited by CVIB was mediated by the N-type VGCC, whereas the irreversibly blocked current (∼ 22% of total) was attributable to P/Q-type VGCCs. ω-Conotoxin CVIB reversibly inhibited Ba2+ currents mediated by N- (CaV2.2) and P/Q- (CaV2.1), but not R- (CaV2.3) type VGCCs expressed in Xenopus oocytes. The α2δ1 auxiliary subunit co-expressed with CaV2.2 and CaV2.1 reduced the sensitivity of VGCCs to CVIB but had no effect on reversibility of block. Determination of the NMR structure of CVIB identified structural differences to CVID that may underlie differences in selectivity of these closely related conotoxins. ω-Conotoxins CVIB and CVID may be useful as antagonists of N- and P/Q-type VGCCs, particularly in sensory neurons involved in processing primary nociceptive information.