Key points Regulation of ion channel function during repeated firing of action potentials is commonly observed in excitable cells. Recently it was shown that muscle activity is associated with rapid, protein kinase C (PKC)‐dependent ClC‐1 Cl− channel inhibition in rodent muscle. While this PKC‐dependent ClC‐1 inhibition during muscle activity was shown to be important for the maintenance of contractile endurance in rat muscle it is unknown whether a similar regulation exists in human muscle. Also, the molecular mechanisms underlying the observed PKC‐dependent ClC‐1 inhibition are unclear. Here we present the first demonstration of ClC‐1 inhibition in active human muscle fibres, and we determine the changes in ClC‐1 gating that underlie the PKC‐dependent ClC‐1 inhibition in active muscle using human ClC‐1 expressed in Xenopus oocytes. This activity‐induced ClC‐1 inhibition is suggested to represent a mechanism by which human muscle fibres maintain their excitability during sustained activity. Abstract Repeated firing of action potentials (APs) is known to trigger rapid, protein kinase C (PKC)‐dependent inhibition of ClC‐1 Cl− ion channels in rodent muscle and this inhibition is important for contractile endurance. It is currently unknown whether similar regulation exists in human muscle, and the molecular mechanisms underlying PKC‐dependent ClC‐1 inhibition are unclear. This study first determined whether PKC‐dependent ClC‐1 inhibition exists in active human muscle, and second, it clarified how PKC alters the gating of human ClC‐1 expressed in Xenopus oocytes. In human abdominal and intercostal muscles, repeated AP firing was associated with 30–60% reduction of ClC‐1 function, which could be completely prevented by PKC inhibition (1 μm GF109203X). The role of the PKC‐dependent ClC‐1 inhibition was evaluated from rheobase currents before and after firing 1000 APs: while rheobase current was well maintained after activity under control conditions it rose dramatically if PKC‐dependent ClC‐1 inhibition had been prevented with the inhibitor. This demonstrates that the ClC‐1 inhibition is important for maintenance of excitability in active human muscle fibres. Oocyte experiments showed that PKC activation lowered the overall open probability of ClC‐1 in the voltage range relevant for AP initiation in muscle fibres. More detailed analysis of this reduction showed that PKC mostly affected the slow gate of ClC‐1. Indeed, there was no effect of PKC activation in C277S mutated ClC‐1 in which the slow gate is effectively locked open. It is concluded that regulation of excitability of active human muscle fibres relies on PKC‐dependent ClC‐1 inhibition via a gating mechanism.