TWIK1 (K2P1.1/KCNK1) belongs to the potassium channels of the two-pore domain. Its current is very small and difficult to measure. In this work, we used a 100 mM NH4+ extracellular solution to increase TWIK1 current in its stable cell line expressed in HEK293. Then, the inhibition of magnolol on TWIK1 was observed via a whole-cell patch clamp experiment, and it was found that magnolol had a significant inhibitory effect on TWIK1 (IC50 = 6.21 ± 0.13 μM). By molecular docking and alanine scanning mutagenesis, the IC50 of TWIK1 mutants G229A, T225A, I140A, L223A, and S224A was 20.77 ± 3.20, 21.81 ± 7.93, 10.22 ± 1.07, 9.55 ± 1.62, and 7.43 ± 3.20 μM, respectively. Thus, we conclude that the inhibition of the TWIK1 channel by magnolol is related to G229 and T225 on the P2- pore helix.