SETD3 is a member of SET-domain containing methyltransferase family, which plays critical roles in various biological events. It has been shown that SETD3 could regulate the transcription of myogenic regulatory genes in C2C12 differentiation and promote myoblast determination. However, how SETD3 is regulated during myoblast differentiation is still unknown. Here, we report that two important microRNAs (miRNAs) could repress SETD3 and negatively contribute to myoblast differentiation. Using microRNA (miRNA) prediction engines, we identify and characterize miR-15b and miR-322 as the primary miRNAs that repress the expression of SETD3 through directly targeting the 3'-untranslated region of SETD3 gene. Functionally, overexpression of miR-15b or miR-322 leads to the repression of endogenous SETD3 expression and the inhibition of myoblast differentiation, whereas inhibition of miR-15b or miR-322 derepresses endogenous SETD3 expression and facilitates myoblast differentiation. In addition, knockdown SETD3 in miR-15b or miR-322 repressed myoblasts is able to rescue the facilitated differentiation phenotype. More interestingly, we revealed that transcription factor E2F1 or FAM3B positively or negatively regulates miR-15b or miR-322 expression, respectively, during muscle cell differentiation, which in turn affects SETD3 expression. Therefore, our results establish two parallel cascade regulatory pathways, in which transcription factors regulate microRNAs fates, thereby controlling SETD3 expression and eventually determining skeletal muscle differentiation.