The plant NADPH oxidases, known as respiratory burst oxidase homologues (Rbohs), play an indispensable role in a wide array of cellular and developmental processes. Arabidopsis thaliana RbohF (AtRbohF)-mediated production of reactive oxygen species (ROS) is involved in biotic and abiotic stress responses. Because of the toxicity of excess amount of ROS, the ROS-producing activity of Rbohs is speculated to be negatively regulated. However, its mechanism is mostly unknown to date. Here, we report the identification of calcineurin B-like protein-interacting protein kinase 26 (CIPK26) as a novel regulatory factor of AtRbohF. We isolated CIPK26 as an AtRbohF-interacting partner by a yeast two-hybrid screen. Our co-immunoprecipitation assay revealed that the CIPK26 protein interacts with the N-terminal region of AtRbohF in Nicotiana benthamiana cell extracts. The fluorescence of both GFP-tagged CIPK26 and AtRbohF was predominantly observed at the cell periphery. We also showed that co-expression of CIPK26 decreases the ROS-producing activity of AtRbohF in HEK293T cells. Together, these results suggest that the direct binding of CIPK26 to AtRbohF negatively modulates ROS production and play a role in the regulation of ROS signalling in plants.