Growth factor withdrawal from proliferating myoblasts induces the expression of muscle-specific genes essential for myogenesis. By suppression subtractive hybridization (SSH), we have cloned a novel human cDNA that encodes a Cys3His zinc finger protein named CHCR (Cys3His CCG1-Required). CHCR is related to Muscleblind (Mbl), a Drosophila melanogaster protein required for terminal muscle differentiation. It also displays sequence similarity to EXP/MBNL, a human Mbl protein that interacts with CUG expansions associated with the degenerative muscular disease, myotonic dystrophy (DM1). This relationship with EXP/MBNL and Mbl suggests that CHCR also functions during muscle differentiation. We have found that CHCR mRNA and protein levels decrease upon differentiation of mouse myoblast cells. Constitutive expression of CHCR in C2C12 cells inhibits the induction of sarcomeric myosin heavy chain (MyHC) upon serum deprivation. Induction of myogenin, an earlier marker of muscle differentiation, is inhibited to a lesser extent, while expression of the cell cycle inhibitor, p21, remains unaffected. Loss of CHCR function by morpholino antisense oligonucleotide treatment accelerates MyHC induction during differentiation of myoblast cells. These complementary gain- and loss-of-function results suggest that CHCR is an inhibitor of myogenesis. CHCR represents the first muscleblind-related protein that antagonizes, instead of promotes, muscle differentiation.