Cyanobacterial DnaJ offers thermo-tolerance and effectively prevents aggregation of denatured protein in coordination with DnaK. The hypothetical protein All3048 of Anabaena sp. PCC7120 was found to be a 24 kDa DnaJ III protein with a putative J-domain at the extreme N-terminus. This paper decodes the role of All3048 in thermo-tolerance and as a co-chaperon of DnaK. Semi-quantitative and RT-PCR results showed up-accumulation of all3048 in heat, UV-B, cadmium, arsenic and salt. BL21/pET-28a-all3048, all3048(1–95) and all3048(31–128) reduced the heat stress-induced ROS generation by 40 %, 21 % and 24 % as compared to BL21/pET-28-a. Conformational properties of All3048 and its truncated variants were assessed using bis ANS, guanidine hydrochloride and acrylamide quenching. All3048(1–95), All3048 and All3048(31–128) increased DnaK ATPase activity by 8.6, 8.2, and 2.5 fold, respectively. The thermostability investigated using DSC and DSF methods affirmed the relative stability of All3048 and All3048 (31–128), whereas All3048 (1–95) was the least stable. All3048 is a novel cyanobacterial DnaJ III that imparts heat stress tolerance in E. coli; however, only the J-domain present at N-terminus was sufficient for stimulating DnaK's ATPase activity.