Root growth and development depend on continuous cell division and differentiation in root tips. In these processes, reactive oxygen species (ROS) play a critical role as signaling molecules. However, few ROS signaling regulators have been identified. In this study, we found knockdown of a syntaxin gene, SYNTAXIN OF PLANTS81 in Arabidopsis thaliana (AtSYP81) resulted in severe reduction in root meristem activity and disruption of root stem cell niche (SCN) identity. Subsequently, we found AtSYP81 was highly expressed in roots and localized on the endoplasmic reticulum (ER). Interestingly, the reduced expression of AtSYP81 conferred decreased number of peroxisomes in root meristem cells, raising a possibility that AtSYP81 regulates root development through peroxisome-mediated ROS production. Further transcriptome analysis revealed that class III peroxidases, which are responsible for intracellular ROS homeostasis, showed significantly changed expression in the atsyp81 mutants and AtSYP81 overexpression lines, adding evidence of the regulatory role of AtSYP81 in ROS signaling. Accordingly, rescuing the decreased ROS level via applying ROS donors effectively restored the defects in root meristem activity and SCN identity in the atsyp81 mutants. APETALA2 (AP2) transcription factors PLETHORA1 and 2 (PLT1 and PLT2) were then established as the downstream effectors in this pathway, while potential crosstalk between ROS signaling and auxin signaling was also indicated. Taken together, our findings suggest that AtSYP81 regulates root meristem activity and maintains root SCN identity by controlling peroxisome- and peroxidase-mediated ROS homeostasis, thus both broadening and deepening our understanding of biological roles of SNARE proteins and ROS signaling.