The actin-binding protein α-actinin-3 is one of the two isoforms of α-actinin that are found in the Z-discs of skeletal muscle. α-Actinin-3 is exclusively expressed in fast glycolytic muscle fibers. Homozygosity for a common polymorphism in the ACTN3 gene results in complete deficiency of α-actinin-3 in about 1 billion individuals worldwide. Recent genetic studies suggest that the absence of α-actinin-3 is detrimental to sprint and power performance in elite athletes and in the general population. In contrast, α-actinin-3 deficiency appears to be beneficial for endurance athletes. To determine the effect of α-actinin-3 deficiency on the contractile properties of skeletal muscle, we studied isolated extensor digitorum longus (fast-twitch) muscles from a specially developed α-actinin-3 knockout (KO) mouse. α-Actinin-3-deficient muscles showed similar levels of damage to wild-type (WT) muscles following lengthening contractions of 20% strain, suggesting that the presence or absence of α-actinin-3 does not significantly influence the mechanical stability of the sarcomere in the mouse. α-Actinin-3 deficiency does not result in any change in myosin heavy chain expression. However, compared with α-actinin-3-positive muscles, α-actinin-3-deficient muscles displayed longer twitch half-relaxation times, better recovery from fatigue, smaller cross-sectional areas, and lower twitch-to-tetanus ratios. We conclude that α-actinin-3 deficiency results in fast-twitch, glycolytic fibers developing slower-twitch, more oxidative properties. These changes in the contractile properties of fast-twitch skeletal muscle from α-actinin-3-deficient individuals would be detrimental to optimal sprint and power performance, but beneficial for endurance performance.