Pannexins are plasma membrane heptameric channels mediating ATP release from the cytosol to the extracellular space. Skeletal muscle activity is associated with Pannexin 1 (Panx1) channels activation, ATP release out to the extracellular space and subsequent activation of purinergic signaling pathways. In agreement, recent evidence has shown molecular and functional interactions between Panx1 and the excitation–contraction (EC) coupling machinery of skeletal muscle. In this framework, we tested whether pharmacological effectors of Panx1 affect EC coupling in differentiated muscle fibers. Using confocal detection of cytosolic Ca²⁺ in voltage-clamped mouse muscle fibers, we found that the Panx1 blocker probenecid (1 mM) affects intracellular Ca²⁺ handling and EC coupling: acute application of probenecid generates a rise in resting Ca²⁺ that also occurs in nominally Ca²⁺-free extracellular medium. This effect is associated with a reduction of Ca²⁺ release through the sarcoplasmic reticulum (SR) Ca²⁺ channel RYR1. The effect of probenecid persists with time, with muscle fibers incubated for 30 min in the presence of the drug exhibiting a 40% reduction in peak SR Ca²⁺ release. Under the same conditions, the other Panx1 blocker carbenoxolone (50 µM) produced a 70% reduction in peak SR Ca²⁺ release. Application of probenecid on electrically stimulated whole mouse muscle induced a slight rise in resting tension and a >50% reduction of tetanic force after 30 min of incubation. Our results provide further support for the strong links between Panx1 function and EC coupling. Because probenecid is used both in the clinic for several types of therapeutic benefits and as a hiding agent for doping in sport, our results question whether potential adverse muscular effects may have, so far, been overlooked.