Strength deficits associated with eccentric contraction-induced muscle injury stem, in part, from impaired voltage-gated sarcoplasmic reticulum (SR) Ca2+ release. FKBP12 is a 12-kD immunophilin known to bind to the SR Ca2+ release channel (ryanodine receptor, RyR1) and plays an important role in excitation-contraction coupling. To assess the effects of eccentric contractions on FKBP12 content, we measured anterior crural muscle (tibialis anterior [TA], extensor digitorum longus [EDL], extensor hallucis longus muscles) strength and FKBP12 content in pellet and supernatant fractions after centrifugation via immunoblotting from mice before and after a single bout of either 150 eccentric or concentric contractions. There were no changes in peak isometric torque or FKBP12 content in TA muscles after concentric contractions. However, FKBP12 content was reduced in the pelleted fraction immediately after eccentric contractions, and increased in the soluble protein fraction 3 day after injury induction. FKBP12 content was correlated (P = 0.025; R2 = 0.38) to strength deficits immediately after injury induction. In summary, eccentric contraction-induced muscle injury is associated with significant alterations in FKBP12 content after injury, and is correlated with changes in peak isometric torque.