Recent studies have established the importance of estrogen-related receptor γ (ERRγ) as a required participant for insulin secretion in pancreatic β cells. Key downstream genes of ERRγ remain unclear in the pancreatic β cell. To understand the molecular role of ERRγ and elucidate potential key candidate genes involved in pancreatic β cells, the eukaryotic expression plasmid containing mouse ERRγ was constructed and transfected into NIT-1 pancreatic β cells. Overexpression of ERRγ was confirmed by quantitative real-time reverse-transcription polymerase chain reaction and Western blot analyses. RNA-seq was conducted to get the gene expression profiling between the overexpression group cells and control cells. Differentially expressed genes (DEGs) were identified by edgR and subsequently analyzed by gene ontology (GO) enrichment analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis. We found that overexpression of ERRγ in pancreatic β cells enables regulation of the expression of certain genes involved in cell apoptosis and mitochondrial function, such as TFPT, Bcl7c, Dap, Thoc6, Ube2d3, ATP5H, MPV17, and NDUFA6. GO analysis revealed that the DEGs were mainly enriched in apoptotic process, cytoplasm, and protein binding. KEGG pathway analysis demonstrated that downregulated DEGs were mainly enriched in protein processing in endoplasmic reticulum, estrogen signaling pathway, and metabolic pathways. This study helps to further understand and reposition the molecular mechanisms of ERRγ in β cells.