Human papillomavirus (HPV) infection is the most common sexually transmitted infection. Vaccines for HPV infection can reduce the risk of developing cervical cancer. To further improve such vaccines and to explore other methods of preventing or treating viral infection, longitudinal studies in experimental animals are desirable. Here, we describe a newly developed multicolor endoscopic fluorescence imaging system to visualize early HPV infection with fluorescent protein-encoded pseudoviruses (PsV) in the female genital tract of living mice. With this imaging method, the course of HPV PsV infection and the effects of intervention to prevent infection can be monitored in a single mouse over time. Female immunocompetent or athymic mice were pretreated with a vaginal spermicide and then HPV PsV composed of an authentic viral capsid and encapsidating green or red fluorescent protein (GFP or RFP) reporter gene was intravaginally instilled. Expression of GFP or RFP was detected 1 day after PsV challenge, which peaked after 2 or 3 days and decreasing thereafter. No fluorescence was detected in vaccine-treated immunocompetent mice. By using serial infection of the same PsV type (HPV16) encoding either GFP or RFP, different infection patterns of repeated exposure can be monitored. This method offers the ability to monitor experimental virus infections before and after intervention, thereby accelerating the development of appropriate prevention and therapy.