A novel mibefradil derivative, NNC55-0396, designed to be hydrolysis-resistant, was shown to be a selective T-type Ca²⁺ channel inhibitor without L-type Ca²⁺ channel efficacy. However, its effects on cytochromes P450 (P450s) have not previously been examined. We investigated the inhibitory effects of NNC55-0396 toward seven major recombinant human P450s—CYP3A4, CYP2D6, CYP1A2, CYP2C9, CYP2C8, CYPC19, and CYP2E1—and compared its effects with those of mibefradil and its hydrolyzed metabolite, Ro40-5966. Our results show that CYP3A4 and CYP2D6 are the two P450s most affected by mibefradil, Ro40-5966, and NNC55-0396. Mibefradil (IC₅₀ = 33 ± 3 nM, K_i = 23 ± 0.5 nM) and Ro40-5966 (IC₅₀ = 30 ± 7.8 nM, K_i = 21 ± 2.8 nM) have a 9- to 10-fold greater inhibitory activity toward recombinant CYP3A4 benzyloxy-4-trifluoromethylcoumarin-O-debenzylation activity than NNC55-0396 (IC₅₀ = 300 ± 30 nM, K_i = 210 ± 6 nM). More dramatically, mibefradil (IC₅₀ = 566 ± 71 nM, K_i = 202 ± 39 nM) shows 19-fold higher inhibition of CYP3A-associated testosterone 6β-hydroxylase activity in human liver microsomes compared with NNC55-0396 (IC₅₀ = 11 ± 1.1 μM, K_i = 3.9 ± 0.4 μM). Loss of testosterone 6β-hydroxylase activity by recombinant CYP3A4 was shown to be time- and concentration-dependent with both compounds. However, NNC55-0396 (K_I = 3.87 μM, K_inact = 0.061/min) is a much less potent mechanism-based inhibitor than mibefradil (K_I = 83 nM, K_inact = 0.048/min). In contrast, NNC55-0396 (IC₅₀ = 29 ± 1.2 nM, K_i = 2.8 ± 0.3 nM) and Ro40-5966 (IC₅₀ = 46 ± 11 nM, K_i = 4.5 ± 0.02 nM) have a 3- to 4-fold greater inhibitory activity toward recombinant CYP2D6 than mibefradil (IC₅₀ = 129 ± 21 nM, K_i = 12.7 ± 0.9 nM). Our results suggest that NNC55–0396 could be a more favorable T-type Ca²⁺ antagonist than its parent compound, mibefradil, which was withdrawn from the market because of strong inhibition of CYP3A4.