The protein encoded by AtDHyPRP1 (DOUBLE HYBRID PROLINE-RICH PROTEIN 1) contains two tandem PRD-8CMs (proline-rich domain-eight cysteine motif) and represents a new type of HyPRPs (hybrid proline-rich proteins). Confocal microscopy to transgenic Arabidopsis plants revealed that AtDHyPRP1-GFP was localised to plasmalemma, especially plasmodesmata. AtDHyPRP1 mainly expressed in leaf tissues and could be induced by salicylic acid, methyl jasmonate, virulent Pseudomonas syringae pv. tomato DC3000 (Pst DC3000) and avirulent P. syringae pv. tomato DC3000 harbouring avrRPM1 (Pst avrRPM1), suggesting it is involved in defence response of Arabidopsis thaliana (L. Heynh.). After treatments with bacterial suspension of virulent Pst DC3000 or conidial suspension of Botrytis cinerea, AtDHyPRP1 overexpressing lines exhibited enhanced resistance, whereas AtDHyPRP1 RNA interference lines became more susceptible to the pathogens with obvious chlorosis or necrosis phenotypes. In systemic acquired resistance (SAR) analyses, distal leaves were challenged with virulent Pst DC3000 after inoculation of the primary leaves with avirulent Pst avrRPM1 (AV) or MgSO4 (MV). Compared with MV, the infection symptoms in systemic leaves of wild-type plants and AtDHyPRP1 overexpressing lines were significantly alleviated in AV treatment, whereas the systemic leaves of AtDHyPRP1 RNAi lines were vulnerable to Pst DC3000, indicating AtDHyPRP1 was functionally associated with SAR.