Heat shock protein (hsp) 56 (hsp56) is an immunophilin that acts as a cofactor with hsp90 and exhibits both peptidyl-prolyl isomerase activity and chaperone activity. Previous studies have shown that the hypertrophic effect of cardiotrophin-1 (CT-1) in cardiac cells is dependent on hsp56 induction. CT-1 activates a number of signal-transduction pathways. Therefore, we sought to determine whether these pathways were also important for hsp56-induced hypertrophy using overexpression with transiently transfected plasmid vectors in rat neonatal cardiomyocytes. Here we show that multiple signalling pathways are involved in hsp56-induced hypertrophy, namely the Janus kinase-signal-transducer and activator of transcription, extracellular signal-regulated protein kinase and PI3-kinase/Akt signalling pathways. Dominant-negative mutants and inhibitors of these pathways were able to block the hypertrophy observed as a result of hsp56 overexpression. However, an inhibitor of the p38 mitogen-activated protein kinase (p38 MAPK) pathway was not able to block the hypertrophic effect of hsp56 overexpression. Furthermore, we show that domains I, II and IV of the hsp56 protein may be involved in its hypertrophic effect. These studies show for the first time that multiple signalling pathways are involved in the hypertrophic effect of hsp56 and that overexpression of hsp56 itself is able to activate the necessary signalling pathways, which induce hypertrophy.