Heat shock proteins 70 (HSP70s) could cooperate with structurally diverse HSP40s (J proteins) to generate diverse chaperone networks in various cellular compartments, performing multiple housekeeping and stress-related functions in the organisms. There are two kinds of chloroplast heat shock protein 70 (cpHsc70-1, cpHsc70-2) and multiple J proteins in the Arabidopsis chloroplasts, while the interaction between cpHsc70s and J proteins and the function of most J proteins are largely unknown. In the present study, we found that AtDJC78 interacts with cpHsc70-1 through its C terminal, according to the results of yeast two-hybrid (Y2H) and bimolecular fluorescence complementation (BiFC). Bioinformatics analysis showed that DJC78 is one of the widespread and highly conserved J proteins in plants, AtDJC78 could be transported into chloroplasts, and the expression of AtDJC78 was significantly up-regulated under heat stress. Furthermore, we found that AtDJC78 may be associated with regulating hydrogen peroxide levels under heat stress in plants. These findings suggest that AtDJC78 is a new cochaperone interacting with cpHsc70-1 in the chloroplasts.