The Hsp70 chaperone system is central to the proteostasis network in the cytosol of human cells. Hsp70 has roles in protein folding, the prevention and clearance of aggregates, and various other biological processes. A range of co-chaperone proteins regulate Hsp70, among these the Hsp40/DNAJ proteins are important activators of Hsp70 function. Stress inducible Hsp70 and its non-inducible form Hsc70 are ubiquitously expressed, and the most abundant DNAJs are DNAJA1 (Hdj2), DNAJA2 and DNAJB1 (Hsp40, Hdj1). Here, the mechanisms of Hsc70/Hsp70 with the DNAJs is reviewed. An overview of biological functions of the major DNAJs is then presented, particularly in the context of protein misfolding diseases.