Background Hyperthermia upregulates DNAJA4, a member of heat shock proteins (HSPs) 40 family, in human keratinocytes and HPV-infected tissue. DNAJA4 deficiency enhances growth arrest induced by hyperthermia. Clusterin (CLU) and phosphorylated ERK (p-ERK) play a role in regulating cell proliferation and apoptosis, under environmental stress. Objectives To examine the downstream molecules and signalling pathways of DNAJA4 and assess their roles in cell cycle and apoptosis of keratinocytes in response to hyperthermia. Methods Wild-type and DNAJA4-knockout (KO) HaCaT cells were exposed to either 44°C (hyperthermia) or 37°C (control) for 30 min. The expression levels of CLU and pERK were determined by RT-PCR and Western blotting. RNAi and PD98059 were used to inhibit the expression of CLU and pERK , respectively. Cell viability, cell cycle and apoptosis were analysed by MTS assay and flow cytometry. Fresh biopsy samples of human normal foreskin or condyloma acuminatum (CA) were utilized to examine the expression of CLU and pERK after ex vivo culture at 44°C. Results The expression of CLU and pERK was significantly increased by hyperthermia treatment at 44°C in HaCaT cells, foreskin and HPV-infected tissues. In HaCaT cells subjected to hyperthermia, DNAJA4 deficiency further augmented the expression of CLU and pERK. CLU deficiency enhanced the pERK expression. Hyperthermia-induced CLU and pERK exerted protective roles mainly through inhibiting apoptosis and maintaining cell cycle, respectively. Conclusions In keratinocytes, CLU and pERK are induced by hyperthermia, an effect which can be further enhanced by DNAJA4 deficiency. CLU deficiency also increases pERK expression. Both CLU and pERK are critical protective factors of human keratinocytes from hyperthermia-induced injury.