| AtTR1, AtCPK28, and AtCPK32 are functioning in the abiotic stress response in Arabidopsis thialiana, and closely associated with the calcium signaling pathway. In order to analyze relationships between AtTR1, AtCPK28 and AtCPK32, we expressed AtTR1, AtCPK28, and AtCPK32 in Escherichia coli, purified expression proteins, and studied the ubiquitination of AtCPK28 and AtCPK32 by AtTR1 in vitro. The results showed that the AtCPK28 and AtCPK32 were multi-ubiquitylated by AtTR1 in vitro. It indicated that the AtCPK28 and AtCPK32 are potential downstream targets of AtTR1 in the regulation of stress resistance signaling pathways. These findings provided experimental evidence to further study the interactions between AtTR1, AtCPK28, and AtCPK32, and also provided some insights of their molecular mechanisms in the plant under abiotic stresses.