Calcium-dependent protein kinases (CDPKs) play critical roles in the regulation of plant growth, development, and stress response. In this study, Camellia sinensis CsCDPK1, CsCDPK3, CsCDPK20, and CsCDPK26 were cloned and functionally characterized. Bioinformatics analyses showed that all analyzed CsCDPK genes encoded the expected CDPK structures. Subcellular localization indicated that the four CsCDPK proteins were localized to the cytoplasm and nucleus in onion epidermal cells. The quantitative real-time PCR results indicated that the four CsCDPK genes exhibited tissue-specific expression patterns. Exposure to heat stress and exogenous abscisic acid induced increases in CsCDPK20 and CsCDPK26 transcript abundance at different time points. Furthermore, overexpression of CsCDPK20 and CsCDPK26 increased the thermotolerance in transgenic Arabidopsis thaliana plants. The proline content in leaves was significantly higher in transgenic plants than that in wild-type under heat stress, whereas malondialdehyde content was lower in transgenic plants. Additionally, expression of stress-responsive genes (i.e., AtAPX1, AtPOD, AtProT1, AtP5CS2, AtHSFA2, AtHSP70, AtHSP101, AtRD29B, AtRAB18, AtABI1, AtRBOHD, and AtRBOHF) was increased in the CsCDPK20 and CsCDPK26 transgenic A. thaliana plants compared with that in the wild type. Collectively, our results suggest that CsCDPK20 and CsCDPK26 may act as positive regulator in C. sinensis response to heat stress.