Background The plant-specific valine-glutamine (VQ) protein family with the conserved motif FxxxVQxLTG reportedly functions with the mitogen-activated protein kinase (MAPK) in plant immunity. However, the roles of VQ proteins in MAPK-mediated resistance to disease in rice remain largely unknown. Results In this study, two rice VQ proteins OsVQ14 and OsVQ32 were newly identified to function as the signaling components of a MAPK cascade, OsMPKK6-OsMPK4, to regulate rice resistance to Xanthomonas oryzae pv. oryzae ( Xoo ). Both OsVQ14 and OsVQ32 positively regulated rice resistance to Xoo . In vitro and in vivo studies revealed that OsVQ14 and OsVQ32 physically interacted with and were phosphorylated by OsMPK4. OsMPK4 was highly phosphorylated in transgenic plants overexpressing OsMPKK6 , which showed enhanced resistance to Xoo . Meanwhile, phosphorylated OsVQ14 and OsVQ32 were also markedly accumulated in OsMPKK6- overexpressing transgenic plants. Conclusions We discovered that OsVQ14 and OsVQ32 functioned as substrates of the OsMPKK6-OsMPK4 cascade to enhance rice resistance to Xoo , thereby defining a more complete signal transduction pathway for induced defenses.