In skeletal muscle differentiation, muscle-specific genes are regulated by two groups of transcription factors, the MyoD and MEF2 families, which work together to drive the differentiation process. Here, we show that ERK5 regulates muscle cell fusion through Klf transcription factors. The inhibition of ERK5 activity suppresses muscle cell fusion with minimal effects on the expression of MyoD, MEF2, and their target genes. Promoter analysis coupled to microarray assay reveals that Klf-binding motifs are highly enriched in the promoter regions of ERK5-dependent upregulated genes. Remarkably, Klf2 and Klf4 expression are also upregulated during differentiation in an ERK5-dependent manner, and knockdown of Klf2 or Klf4 specifically suppresses muscle cell fusion. Moreover, we show that Sp1 transcription factor links ERK5 to Klf2/4, and that nephronectin, a Klf transcriptional target, is involved in muscle cell fusion. Therefore, an ERK5/Sp1/Klf module plays a key role in the fusion process during skeletal muscle differentiation.