Objective To investigate the effect of E5 protein of human papillomavirus type 16 (HPV16 E5) on keratinocyte growth factor-triggered autophagy. Methods Firstly, pCI-neo-E5 recombinant plasmid was constructed. After that, the recombinant plasmid harboring pCI-neo-E5 was transfected into HaCaT cells using LipofectamineTM2000 reagent, and the mRNA transcriptional level of 16 E5 was evaluated by quantitative real-time PCR (qRT-PCR). Then, the expression of LC3BIIwas detected by Western blotting and the localization of LC3B was observed by immunofluorescence in pCI-neo-E5-HA-transfected HaCaT cells triggered with keratinocyte growth factor. Finally, the mRNA transcriptional levels of several autophagy-associated proteins including beclin1, ATG5, ATG7 and LC3B were assayed by qRT-PCR. Results pCI-neo-E5 recombinant plasmid was constructed successfully. After transfection of pCI-neo-E5, a significant increase of HPV16 E5 mRNA was found in HaCaT cells. HPV16 E5 oncoprotein obviously reduced the expression level of LC3BII and the amount of LC3B puncta in HaCaT cells stimulated with keratinocyte growth factor, and inhibited the mRNA transcription of several autophagy-associated proteins. Conclusion HPV16 E5 can suppress keratinocyte growth factor-triggered autophagy in human keratinocytes through down-regulating autophagy-associated protein expression.