The aim of this study was to enhance the proliferation efficiency of spermatogonial stem cells (SSCs). In order to improve the proliferation efficiency, we investigated new factors that promote the proliferation of SSCs using in vitro culture method with natural plant extracts. Germ cell populations containing SSCs were collected 6- to 8-days-old from C57BL/6-TG-EGFP (C57GFP) mice and SSCs were isolated from the collected cells via magnetic-activated cell sorting (MACS). Since then, SSCs were cultured for a week with culture medium containing natural plant extracts at concentration of 0.1, 1, and 10 μg/mL. After a week of culture, we looked for an increase, especially a dose-dependent increase, in the number of cells compared to that of the control group. A dose-dependent increase, in the number of cells was observed in the Petasides japonicus-treated groups. Furthermore, we carried out repeated experiment that is process consisting of selection and additional segmentation to explore new factors for activating SSCs at the molecular level. As a results, Petasides japonicus butanol fraction significantly increased the proliferation rate of SSCs in a dose-dependent manner among Petasides japonicus fraction samples. We identified normal expression level of PLZF in SSCs cultured with plant extracts using immunocytochemistry method. Furthermore, we also carried out qRT-PCR and identified normal expression level of Lhx1 and GFRα1. The finding of this study could contribute to improvement of proliferation and activation for SSCs, using culture method with natural plant extracts.