Human papillomavirus (HPV) is the most common sexually transmitted pathogen in the United States, causing 99% of cervical cancers and 5% of all human cancers worldwide. HPV infection requires transport of the viral genome (vDNA) into the nucleus of basal keratinocytes. During this process, minor capsid protein L2 facilitates subcellular retrograde trafficking of the vDNA from endosomes to the Golgi, and accumulation at host chromosomes during mitosis for nuclear retention and localization during interphase. Here we investigated the relationship between cellular glutathione (GSH) and HPV16 infection. siRNA knockdown of GSH biosynthetic enzymes results in a partial decrease of HPV16 infection. Likewise, infection of HPV16 in GSH depleted keratinocytes is inefficient, an effect that was not seen with adenoviral vectors. Analysis of trafficking revealed no defects in cellular binding, entry, furin cleavage of L2, or retrograde trafficking of HPV16, but GSH depletion hindered post-Golgi trafficking and translocation, decreasing nuclear accumulation of vDNA. Although precise mechanisms have yet to be defined, this work suggests that GSH is required for a specific post-Golgi trafficking step in HPV16 infection.