To study the cellular and molecular function of peroxisome proliferator-activated receptor gamma (PPAR gamma) in skeletal muscle differentiation, we have generated inducible gain-of-function to overexpress PPAR gamma in C2C12 myoblasts. In order to identify PPAR gamma targets, RNA sequencing (RNA-seq) was used to evaluate and quantify the transcriptomes and expression patterns during myogenic differentiation under the overexpression of PPAR gamma. The formation of myotubes and the expression of muscle-specific myogenic genes such as MyoD and MyoG may be inhibited by PPAR gamma overexpression. Multiple genes and pathways were significantly involved in this process, including 11 genes such as Fndc9 and Slc14a1 with fundamental change of regulation modes, 9 genes of which were validated by the data of qRT-PCR. Our studies demonstrate that PPAR gamma would play critical roles on myoblasts differentiation, mediating crosstalk among several pathways and transcription factors. Our data is available in the Gene Expression Omnibus (GEO) database with the accession number as GSE99399.