Activation of receptor for advanced glycation end products (RAGE) by its ligand, HMGB1, stimulates myogenesis via a Cdc42-Rac1-MKK6-p38 mitogen-activated protein kinase pathway. In addition, functional inactivation of RAGE in myoblasts results in reduced myogenesis, increased proliferation, and tumor formation in vivo. We show here that TE671 rhabdomyosarcoma cells, which do not express RAGE, can be induced to differentiate on transfection with RAGE (TE671/RAGE cells) but not a signaling-deficient RAGE mutant (RAGEDeltacyto) (TE671/RAGEDeltacyto cells) via activation of a Cdc42-Rac1-MKK6-p38 pathway and that TE671/RAGE cell differentiation depends on RAGE engagement by HMGB1. TE671/RAGE cells also show p38-dependent inactivation of extracellular signal-regulated kinases 1 and 2 and c-Jun NH(2) terminal protein kinase and reduced proliferation, migration, and invasiveness and increased apoptosis, volume, and adhesiveness in vitro; they also grow smaller tumors and show a lower tumor incidence in vivo compared with wild-type cells. Two other rhabdomyosarcoma cell lines that express RAGE, CCA and RMZ-RC2, show an inverse relationship between the level of RAGE expression and invasiveness in vitro and exhibit reduced myogenic potential and enhanced invasive properties in vitro when transfected with RAGEDeltacyto. The rhabdomyosarcoma cell lines used here and C2C12 myoblasts express and release HMGB1, which activates RAGE in an autocrine manner. These data suggest that deregulation of RAGE expression in myoblasts might concur in rhabdomyosarcomagenesis and that increasing RAGE expression in rhabdomyosarcoma cells might reduce their tumor potential.