The oncoprotein E6 of high-risk human papillomavirus (HPV) is responsible for the initiation and progression of cervical cancer. In the present study， we performed RNA-sequencing analysis in HPV16 E6-expressing 293T cells and identified 56 differentially expressed genes (DEGs) and a number of cellular pathways significantly affected. We confirmed nine of these DEGs in both cell models and clinical tissue samples. Furthermore， we performed de novo transcriptome assembly of 1128 novel human transcripts and identified 22 that are differentially expressed in the presence of HPV16 E6. In addition， our analysis revealed distinct alternative splicing events in response to HPV16 E6 expression. Overall， the present study provides a comprehensive portrait of transcriptional and splicing signatures， as well as previously unknown genes differentially expressed in response to HPV16 E6， which prompts the need for a complete annotation of the HPV16 E6-regulated transcriptome.