We developed a yeast cell-free system suitable for in vitro translation of human papillomavirus 58 (HPV58) L1 mRNA. This system was systematically optimized resulting in enhanced translation efficiency. The optimal concentrations of potassium and magnesium ions observed were specific to the HPV58 L1 protein production. Supplementation with sucrose in the preparation of the yeast lysate greatly enhanced its stability. After optimization, protein production in this system was significantly superior to that produced by the rabbit reticulocyte (RRL) system. Finally, we demonstrated for the first time that virus-like particles (VLPs) were assembled from HPV58 L1 capsid protein in the yeast cell-free system. Thus, the system described here is a powerful tool for the HPV L1 protein production and will be useful for the study of VLP assembly and DNA encapsulation.