Orphan receptors comprise nearly half of all members of the nuclear receptor superfamily. Despite having broad structural similarities to the classical estrogen receptors, estrogen-related receptors (ERRs) have their own unique DNA response elements and functions. In this study, we focus on 2 ERRbeta splice variants, short form ERRbeta (ERRbetasf) and ERRbeta2, and identify their differing roles in cell cycle regulation. Using DY131 (a synthetic agonist of ERRbeta), splice-variant selective shRNA, and exogenous ERRbetasf and ERRbeta2 cDNAs, we demonstrate the role of ERRbetasf in mediating the G1 checkpoint through p21. We also show ERRbetasf is required for DY131-induced cellular senescence. A key novel finding of this study is that ERRbeta2 can mediate a G2/M arrest in response to DY131. In the absence of ERRbeta2, the DY131-induced G2/M arrest is reversed, and this is accompanied by p21 induction and a G1 arrest. This study illustrates novel functions for ERRbeta splice variants and provides evidence for splice variant interaction.