The delivery of small interfering RNA (siRNA) is an attractive therapeutic approach to treat several pathologies, such as viral infections or cancers. However, the stability and the efficacy of these biotherapies are still a major obstacle to their use. Cationic liposomes (DOTAP/Chol/DOPE 1/0.75/0.5 M ratio) have been complexed to siRNA (lipoplexes) in order to be administrated by the vaginal route, in the context of HPV16 induced cervical preneoplastic lesions. To overcome the constraint of the cervico-vaginal mucus, PEGylation is required to allow the diffusion of lipoplexes through it. Thereby, PEGylated lipoplexes coated with three types of polyethylene glycol (PEG) as DSPE-PEG2000, DSPE-PEG750 or C8-PEG2000-Ceramide (Ceramide-PEG2000) at different densities have been developed and characterized. PEGylated lipoplexes were successfully prepared and showed a hydrodynamic diameter around 200 nm, appropriate for vaginal application. In vitro assays on HPV16 positive cell lines revealed that a positive charge of PEGylated lipoplexes allows a higher mRNA knockdown by siRNA. However, the cationic property is also associated to cytotoxicity. The addition of a high percentage of PEG prevented this toxicity but seemed also to reduce siRNA endosomal escape, probably by steric hindrance. The decreasing of PEG density of Ceramide-PEG2000 to 20% allows the release of siRNA and in consequence, biological activities, contrarily to DSPE-PEG. These results suggest that Ceramide-PEG is more appropriate for siRNA delivery compared to DSPE-PEG. In conclusion, the right balance between cytotoxicity and siRNA effectiveness has been found with the transfection of lipoplexes coated with 20% of Ceramide-PEG2000. This new nanovector could have a high potential against multiple mucosal diseases, such as human papillomavirus-induced genital lesions.