Potassium transport channels regulate cellular excitability and membrane potentials. Dysfunction of potassium channels may happen under certain pathophysiological conditions via post-translational modification (PTM), inducing the S-glutathionylation, in oxidative stress. Here, we try to demonstrate the effect of ROS on Kir4.1 and Kir5.1 channel. It is found that the target subunit of ROS is Kir5.1 instead of Kir4.1 subunit. Kir4.1-Kir5.1 heteromeric channel is inhibited by different kinds of oxidants. The patch clamp study confirmed that these oxidants work on the intracellular side of the Kir5.1 subunit. Further experiments demonstrate that the inhibition of Kir4.1-Kir5.1 channels is through S-glutathionylation of the Kir5.1 subunit on Cys158. The information we find may help to better understand the Kir4.1 and Kir5.1 channel modulation mechanism in various pathophysiological conditions, such as oxidative stress.