Muscle disuse produced by hindlimb unloading (HU) induces severe atrophy and slow-to-fast fibre type transition of the slow-twitch soleus muscle (Sol). After 2 weeks HU, the resting ClC-1 chloride conductance (g(Cl)) of sarcolemma, which controls muscle excitability, increases in Sol toward a value typical of the fast-twitch EDL muscle. After 3 days of HU, the g(Cl) increases as well before initiation of fibre type transition. Since ClC-1 channels are acutely silenced by PKC-dependent phosphorylation, we studied the modulation of g(Cl) by PKC and serine-threonine phosphatase in Sol during HU, using a number of pharmacological tools. We show that a fraction of ClC-1 channels of control Sol are maintained in an inactive state by PKC basal activity, which contributes to the lower g(Cl) in control Sol compared to EDL. After 14 days of HU, PKC/phosphatase manipulation produces effects on Sol g(Cl) that corroborate the partial slow-to-fast transition. After 3 days of HU, the early increase of g(Cl) in Sol is entirely attributable to a reduction of PKC activity and/or activation of phosphatase, maintaining ClC-1 channels in a fully active state. Accordingly, we found that HU reduces expression of PKCalpha, epsilon, and isoenzymes in Sol and EDL muscles and reduces total PKC activity. Moreover, we show that the rheobase current is increased in Sol muscle fibres as soon as after 3 days of HU, most probably in relation to the increased g(Cl). In conclusion, Sol muscle disuse is characterized by a rapid reduction of PKC activity, which reduces muscle excitability and is likely to contribute to disuse-induced muscle impairment.