Thioredoxin (Trx) is a key player in redox homeostasis in various cells, modulating the functions of target proteins by catalyzing a thiol–disulfide exchange reaction. Target proteins of cytosolic Trx‐h of higher plants were studied, particularly in the plasma membrane, because plant plasma membranes include various functionally important protein molecules such as transporters and signal receptors. Plasma membrane proteins from Arabidopsis thaliana cell cultures were screened using a resin Trx‐h1 mutant‐immobilized, and a total of 48 candidate proteins obtained. These included two calcium‐sensing proteins: a phosphoinositide‐specific phospholipase 2 (AtPLC2) and a calcium‐dependent protein kinase 21 (AtCPK21). A redox‐dependent change in AtCPK21 kinase activity was demonstrated in vitro. Oxidation of AtCPK21 resulted in a decrease in kinase activity to 19% of that of untreated AtCPK21, but Trx‐h1 effectively restored the activity to 90%. An intramolecular disulfide bond (Cys97–Cys108) that is responsible for this redox modulation was then identified. In addition, endogenous AtCPK21 was shown to be oxidized in vivo when the culture cells were treated with H2O2. These results suggest that redox regulation of AtCPK21 by Trx‐h in response to external stimuli is important for appropriate cellular responses. The relationship between the redox regulation system and Ca2+ signaling pathways is discussed.Structured digital abstract CPK21 physically interacts with TRXH1 by two hybrid (View interaction) TRXH1 physically interacts with AT1G56075, CPK21, PRX2B, HIR3, RAA4B, TC132, RAD2C, AT3G03270, AT3G53990, METK4, AT3G17020, AT1G20760, MPCP3, SKS2, AT5G23890, DTC, AT1G56070, TBB3, ACT8, TI110, DHSB1, TBA3, AOX1A, PLCD2, CLAH1, CLAH2, RSSA1, APX1, RAB1C, ACT7, ARF1, NRL1, ADT1, TBB7, RAD2A, G3PC1, TBB4, MD37E, DHSA1, TC159, AT4G22710, GPX6, RABC1, VATA, GPX2 and RAA2A by affinity chromatography technology (View interaction)