Objective Whether porcine circovirus type 2 (PCV2) can infect human cells is controversial. To study the antiviral effects of human cells on PCV2 and other viruses, we analyzed differentially expressed genes (DEGs) and their related signaling pathways and biological functions in PCV2-infected human cells by transcriptome analysis (RNA sequencing, RNA-seq). Methods HeLa cells were infected with PCV2, and then analyzed by RNA-seq to screen DEGs related to antiviral reactions. The DEGs were further verified by Real-time quantitative PCR. Results A total of 15402 UniGenes and 387 DEGs, including 267 upregulated genes and 120 downregulated genes, were annotated in human HeLa cells infected with PCV2. The most abundant Gene Ontology (GO) terms were response to stress ( 97 / 291 DEGs) and immune system process (80 / 291 DEGs). The most significant pathways were the nucleotide-binding and oligomerization domain ( NOD)-like receptor signaling pathway ( 21 / 170 DEGs ) and herpes simplex infection ( 22 / 170 DEGs ). Conclusions The gene expression profile of HeLa cells significantly changed after PCV2 infection. The result obtained in this study will help understand the antiviral mechanism of human cells and may provide a basis for understanding the immune response and antiviral efficacy of human cells infected with PCV2.