In contrast to fast-twitch skeletal muscle fibers of the chicken, slow-twitch fibers are fatigue-resistant. In fast fibers, the fatigue process has been related to K~ATP~ channels. In the present study, we investigated the action of glibenclamide (an anti-diabetic sulphonylurea that acts on K~ATP~ channels) on fatigued slow skeletal muscle, studying twitch and tetanus tension after inducing the muscle to fatigue by continuous electrical stimulation. Our results showed that glibenclamide (150 μM) increased post-fatigue twitch tension by about 25% with respect to the fatigued condition (P < 0.05). In addition, glibenclamide (150 μM) increased post-fatigue tetanic tension (83.61 ± 15.7% in peak tension, and 85.0 ± 19.0% in tension-time integral, P = 0.02, and 0.04, respectively; n = 3). Moreover, after exposing the muscle to a condition that inhibits mitochondrial ATP formation in order to activate K~ATP~ channels with cyanide (10 mM), tension also diminished, but in the presence of glibenclamide the effect produced by cyanide was abolished. To determine a possible increase in intracellular calcium concentration, the effects of glibenclamide on caffeine-evoked contractures were explored. After muscle pre-incubation with glibenclamide (150 μM), tension of caffeine-evoked contractures increased (6.5 ± 1.5% in maximal tension, and 5.9 ± 3.8% in tension-time integral, P < 0.05). These results suggest a possible role of K~ATP~ channels in the fatigue process, since glibenclamide increases twitch and tetanus tension in fatigued slow muscle of the chicken and during metabolic inhibition, possibly by increasing intracellular calcium.