Abstract Background Due to the potential of human papillomavirus (HPV) vaccination for decreasing cervical cancer rates in Mainland China, where some of the highest incidences in the world have been reported, our study aimed to assess HPV and HPV vaccine knowledge, and to evaluate the effect of a brief educational intervention on HPV knowledge and vaccine acceptability in Chinese undergraduate students and employed women. Methods This multi-center, cross-sectional study was conducted across five representative cities of the five main geographical regions of Mainland China. Participants were selected from one comprehensive university and three to four companies in each city for a total of six comprehensive universities and 16 companies. A 62-item questionnaire on HPV knowledge and HPV vaccine acceptability was administered to participants before and after an educational intervention. The intervention consisted of an informative group lecture. Results A total of 1146 employed women and 557 female undergraduate students were surveyed between August and November 2011. Baseline HPV knowledge was low among both groups— 320/1146 (28%) of employed women and 66/557 (12%) of students had heard of HPV, while only 237/1146 (21%) of employed women and 40/557 (7.2%) of students knew that HPV is related to cervical cancer. After educational instruction, 947/1061 (89%) of employed women and 193/325 (59%) of students knew the relationship between HPV and cervical cancer (χ2 = 1041.8, p < 0.001 and χ2 = 278.5, p < 0.001, respectively). Post-intervention, vaccine acceptability increased from 881/1146 (77%) to 953/1061 (90%), (p = <0.001) in employed women and 405/557 (73%) in students to 266/325 (82%), (p < 0.001). Women in both groups cited concerns about the HPV vaccine’s safety, efficacy, and limited use to date as reasons for being unwilling to receive vaccination. 502/1146 (44%) of women were willing to vaccinate their children at baseline, which increased to 857/1061 (81%) post-intervention, p < 0.001. Conclusions Incorporation of our lecture-based education initiative into a government-sponsored or school-based program may improve HPV-related knowledge and HPV vaccine acceptability. Further studies are needed to evaluate and standardize HPV education programs in China.