The chaperone network plays an essential role in cellular protein homeostasis. However, some core components often coaggregate with misfolded proteins for sequestration and dysfunction, leading to abnormal cell proteostasis, aggregation-associated disorders, and poor solubility of overexpressed recombinant proteins. Among them, DnaJ or its ortholog, an obligate co-chaperone in the tripartite DnaK-DnaJ-GrpE system, is of more implications, probably due to its intrinsic propensity for aggregation. Herein, we potentiated the activity of Escherichia coli DnaJ by using hyper-acidified protein fusion strategy. We found DnaJ did possess only a moderate solubility that could be remarkably improved by fusing hyper-acidic minipeptides. Most importantly, we revealed the hyper-acidified DnaJ with a fusion tail could outperform its native form (significantly up to 2.1-fold) to enhance the solubility of target proteins and meanwhile appropriately impart them an elevated activity. These results suggest the hyper-acidified DnaJs can chaperone target proteins with correct folding into a truly soluble and active form. Moreover, we showed these hyper-acidified DnaJ variants could surpass its prototype to confer E. coli or yeast an enhanced heat tolerance, and DnaJ itself could be solubilized by its hyper-acidified fusion cognates. Finally, we discussed the overall mechanism for DnaJ activity potentiation mediated by hyper-acidic tailing fusion.