Sox6, a member of the Sox family of transcription factors, is highly expressed in skeletal muscle. Despite its abundant expression, the role of Sox6 in muscle development is not well understood. We hypothesize that, in fetal muscle, Sox6 functions as a repressor of slow fiber type‐specific genes. In the wild‐type mouse, differentiation of fast and slow fibers becomes apparent during late fetal stages (after approximately embryonic day 16). However, in the Sox6 null‐p^100H mutant mouse, all fetal muscle fibers maintain slow fiber characteristics, as evidenced by expression of the slow myosin heavy chain MyHC‐β. Knockdown of Sox6 expression in wild‐type myotubes results in a significant increase in MyHC‐β expression, supporting our hypothesis. Analysis of the MyHC‐β promoter revealed a Sox consensus sequence that likely functions as a negative cis‐regulatory element. Together, our results suggest that Sox6 plays a critical role in the fiber type differentiation of fetal skeletal muscle. Developmental Dynamics 236:2062–2076, 2007. © 2007 Wiley‐Liss, Inc.