MyomiRs-IN-1 is a myomiRs inhibitor with activity to inhibit myoD translation in C2C12 cells. MyomiRs-IN-1 does not change the expression level of myoD mRNA , while downregulating the expression of differentiation markers. MyomiRs-IN-1 affects the expression of myomiRs in muscle cells by regulating the regulatory pathway between miR-221/222 and myoD. The application of MyomiRs-IN-1 can facilitate research on muscle development and related diseases.