This experiment was performed to rapidly obtain fibroblasts from guinea pig’s ears, and provide initiating cells for somatic cell reprogramming. The ear tissues were minced and digested with 0.05% Trypsin-EDTA and Collagenase IV. The cells were cultured in vitro and morphologically observed. The cells were detected for mycoplasma and vimentin protein immunofluorescence was performed for characterization. The cell growth curve was drawn to calculate the cell double time. And the infection efficiency was detected by flow cytometry when the cells were infected with retrovirus containing green fluorescent protein. The influence of the virus on the karyotype was detected by karyotype identification. The isolated guinea pig ear fibroblasts were adherent growth cells. Most of them were adherent and fully extended 2~3 hours after digestion. The cells were fusiform or polygonal, with full and stereoscopic cytoplasm. And the nucleus was clear and in good shape. The mycoplasma detection was negative. The cells expressed fibroblast-specific protein vimentin. The growth curve was S-shaped, which was consistent with the normal growth and proliferation rules of cultured cells in vitro. The cell double time was 24.85 h. The efficiency of retrovirus infection was more than 70%, and the cells with chromosome 2n=64 accounted for 90% after viral infection, with genetic stability and normal karyotype. The method is simple and efficient, producing rapidly proliferated cells in good condition. It is an appropriate method for isolating fibroblasts from guinea pig’s ears. The cell line has a high virus infection rate, which can be used for somatic cell reprogramming studies.