The human papillomavirus (HPV) infection may be associated with the development and progression of non-small cell lung cancer (NSCLC). However, the role of HPV-16 oncoproteins in the development and progression of NSCLC is not completely clear. Epithelial-mesenchymal transition (EMT), a crucial step for invasion and metastasis, plays a key role in the development and progression of NSCLC. Here we explored the effect of HPV-16 oncoproteins on EMT and the underlying mechanisms. NSCLC cell lines, A549 and NCI-H460, were transiently transfected with the EGFP-N1-HPV-16 E6 or E7 plasmid. Real-time PCR and Western blot analysis were performed to analyze the expression of EMT markers. A protein microarray was used to screen the involved signaling pathway. Our results showed that overexpression of HPV-16 E6 and E7 oncoproteins in NSCLC cells significantly promoted EMT-like morphologic changes, downregulated the mRNA and protein levels of EMT epithelial markers (E-cadherin and ZO-1), and upregulated the mRNA and protein levels of EMT mesenchymal markers (N-cadherin and vimentin) and transcription factors (ZEB-1 and Snail-1). Furthermore, the HPV-16 E6 oncoprotein promoted STAT3 activation. Moreover, WP1066, a specific signal transducer and activator of transcription 3 (STAT3) inhibitor, reversed the effect of HPV-16 E6 on the expression of ZO-1, vimentin, and ZEB-1 in transfected NSCLC cells. Taken together, our results suggest that overexpression of HPV-16 E6 and E7 oncoproteins enhances EMT, and the STAT3 signaling pathway may be involved in HPV-16 E6-induced EMT in NSCLC cells.