Myosin Binding Protein-C (MyBP-C) consists of three isoforms: slow-skeletal, fast-skeletal, and cardiac. In the present study, our objective is to elucidate the role of fast-skeletal Myosin Binding Protein-C (fsMyBP-C). Using recombinant MyBP-C N-termini, we have previously demonstrated that the fast-skeletal isoform regulates muscle contraction more similarly to cardiac vs. slow-skeletal MyBP-C. However, the physiological implications of such regulation in skeletal muscle remains unclear. Therefore, we generated homozygous knock-out mice that do not express fsMyBP-C in either alleles, as well as heterozygous mice that express fsMyBP-C in only one allele. Currently, no myopathies are associated with mutations in fsMyBP-C, fsMyBP-C expression is dysregulated in skeletal muscle diseases, such as Duchenne Muscular Dystrophy. Both homozygous and heterozygous mice are viable and do not currently exhibit differences in longevity. However, expression of fsMyBP-C are drastically different: homozygous mice do not express fsMyBP-C, but heterozygous mice exhibit the same pattern of expression in skeletal muscle as wild-type (WT) mice. In WT mice, fsMyBP-C is expressed at levels comparable to slow-skeletal MyBP-C in the extensor digitalis longus (EDL) and tibialis anterior (TA) (fast-type and mixed type skeletal muscles, respectively). Interestingly, small amounts of fsMyBP-C are also expressed in the soleus, a slow-type muscle. To analyze functional changes in EDL and soleus muscles, we used Force-ATPase experiments to analyze steady-state myofilament properties, such as force generation, Ca2+-sensitivity, and tension cost. No significant functional changes were observed in heterozygous mice, nor were they observed in the soleus muscle. However, functional deficits became apparent in EDL muscles of homozygous mice, but could be recovered by the addition of recombinant fsMyBP-C N-termini. We propose that homozygous mice may be a useful model for isolating the effects of fsMyBP-C in health and disease.