Human papillomavirus (HPV) virus-like particle (VLP) vaccines were recently licensed. Although neutralizing Ab titers are thought to be the main effectors of protection against infection, early predictors of long-term efficacy are not yet defined and a comprehensive understanding of innate and adaptive immune responses to vaccination is still lacking. Here, microarrays were used to compare the gene expression signature in HPV-16 L1 VLP-stimulated PBMCs from 17 vaccine and 4 placebo recipients before vaccination and 1 mo after receiving the second immunization. Vaccination with a monovalent HPV-16 L1 VLP vaccine was associated with modulation of genes involved in the inflammatory/defense response, cytokine, IFN, and cell cycle pathways in VLP-stimulated PBMCs. Additionally, there was up-regulation of probesets associated with cytotoxic (GZMB, TNFSF10) and regulatory (INDO, CTLA4) activities. The strongest correlations with neutralizing Ab titers were found for cyclin D2 (CCND2) and galectin (LGALS2). Twenty-two differentially expressed probesets were selected for confirmation by RT-PCR in an independent sample set. Agreement with microarray data was seen for more than two-thirds of these probesets. Up-regulation of immune/defense response genes by HPV-16 L1 VLP, in particular, IFN-induced genes, was observed in PBMCs collected before vaccination, with many of these genes being further induced following vaccination. In conclusion, we identified important innate and adaptive response-related genes induced by vaccination with HPV-16 L1 VLP. Further studies are needed to identify gene expression signatures of immunogenicity and long-term protection with potential utility in prediction of long-term HPV vaccination outcomes in clinical trials.