Myostatin (Mstn) is a secreted growth factor belonging to the TGF beta super-family. Inactivation of murine Mstn by gene targeting, or natural mutation of bovine or human Mstn, induces the double muscling (DM) phenotype. In DM cattle, Mstn deficiency increases fast glycolytic (type IIB) fibre formation in biceps femoris (BF) muscle. Using Mstn null (^-/-) mice, we suggest a mechanism behind Mstn mediated fibre type diversity. Histological analysis revealed increased type IIB fibres with a concomitant decrease in type IIA and type I fibres in Mstn^-/- tibialis anterior (TA) and BF muscle. Functional electrical stimulation of Mstn^-/- null BF, revealed increased fatigue susceptibility, supporting increased type IIB fibre content. Given the role of Mef2 in oxidative type I fibre formation, MEF2 levels in Mstn^-/- tissue were quantified. Results revealed reduced MEF2C protein in Mstn^-/- muscle and myoblast nuclear extracts. Reduced MEF2-DNA complex was also observed in EMSA using Mstn^-/- nuclear extracts. Furthermore, reduced expression of MEF2 downstream target genes MLC1F and calcineurin were found in Mstn^-/- muscle. Conversely, Mstn addition was sufficient to directly up-regulate MLC promoter-enhancer activity in cultured myoblasts. Since high MyoD levels are seen in fast fibres and MyoD regulates gene expression in fast glycolytic fibres, we analised MyoD levels. In contrast to MEF2C, MyoD levels were increased in Mstn^-/- muscle. Together, these results suggest that while Mstn positively regulates MEF2C levels, it negatively regulates MyoD expression in muscle. We propose that Mstn alters fibre type composition by regulating the expression of MEF2C and MyoD during myogenesis.