Reactive oxygen species (ROS) play important roles in plant growth, development, responses to abiotic and biotic stresses. Hypersensitive response (HR)-like cell death is often associated with excess ROS. However, how a calcium-dependent protein kinase (CPK) modulates this process remains elusive in rapeseed (Brassica napus L.). In the present study, we identified and characterized CPK6L from rapeseed as a novel regulator of ROS and cell death. The subcellular localization of BnaCPK6L was investigated through GFP and was found to be located at the endoplasmic reticulum membrane. Overexpression of the constitutively active BnaCPK6LCA resulted in significant accumulation of ROS and HR-like cell death than the full-length. A quantitative RT-PCR survey identified that the expression levels of a few ROS, cell death and defense-related marker genes were up-regulated upon BnaCPK6LCA expression. Mating-based split ubiquitin system (mbSUS) screening revealed that BnaCPK6L interacted with BnaRBOHD (Respiratory Burst Oxidase Homolog D), which was validated by bimolecular fluorescence complementation (BiFC). An in vitro phosphorylation assay indicated that BnaCPK6L phosphorylated BnaRBOHD. Lastly, we also found that three 2C type protein phosphatases (PP2Cs) interacted with BnaCPK6L. Taken together, this study indicates that BnaCPK6L plays an important role in ROS and HR-like cell death through interacting with and phosphorylating RBOHD. (C) 2019 Elsevier Inc. All rights reserved.