•A gel containing 3% carrageenan (CG) provides potent and durable protection from HPV infection in mice.•Seminal plasma does not interfere with the in vivo antiviral activity of CG against HPV.•Physicochemical properties of a CG gel formulation influence the in vivo protection against HPV. Commercial vaccines against human papillomavirus (HPV) have low uptake due to parental autonomy, dosing regimen, cost, and cold chain storage requirements. Carrageenan (CG)-based formulations prevent HPV infection in vitro and in vivo but data are needed on the durability of anti-HPV activity and the effect of seminal plasma (SP). The Population Council’s PC-515 gel and the lubricant Divine 9 were tested for their physicochemical properties and anti-HPV activity against HPV16, 18, and 45 pseudoviruses (PsVs). Anti-PsV activity was estimated using the luciferase assay in HeLa cells and the HPV PsV luciferase mouse model. Formulations were applied intravaginally either 2h pre/2h post (−2h/+2h) or 24h pre (−24h) relative to challenge with HPV16 or 45 PsV in PBS or SP/PBS. Both formulations showed broad-spectrum anti-HPV activity in vitro (IC50: 1–20ng/ml), significantly decreasing HPV PsV infection in the mouse model (−2h/+2h, p<0.0001). PC-515 protected better than Divine 9 in the −24h dosing regimen (p<0.0001) and comparable to Divine 9 in the −2h/+2h regimen (p=0.9841). PC-515 retained full activity in the murine model when PsV solutions contained human SP. The durable, potential broad-spectrum anti-HPV activity of CG formulations in the presence of SP supports their further development to prevent HPV acquisition.