In this study, we evaluated the effects of ascorbic acid (VC), epidermal growth factor (EGF) and follicle stimulating hormone (FSH) on in vitro culture of sheep ovarian cortical tissue. Using 2×2×2 factor experimental design, we cultured sheep ovarian cortex fragments in 8 media with MEM (control), MEM+VC (50 μg/mL), MEM +EGF (100 ng/mL), MEM+FSH (50 ng/mL), MEM+VC+EGF, MEM+VC+FSH, MEM+EGF+FSH, MEM+VC+EGF+FSH. After 0 (non-cultured control), 2, 6, 12 days of culture, the pieces of ovarian cortex were proceed to histological and proliferating cell nuclear antigen (PCNA) examination, or observed by transmission electron microscopy (TEM). The percentages of developing follicles were increased (P<0.05) and the percentages of healthy follicles were reduced (P<0.05). When compared to the MEM group, the addition of FSH with VC or EGF promoted a significant increase of follicles diameter and follicles survival rate (P<0.05), and stimulated the proliferation of granulosa cells. After 12 days of culture, medium supplemented with MEM+VC+EGF resulted the lowest proportion of developing follicles (49.3%±3.2%), follicles diameter((32.3±2.3) μm), follicles survival rate (41.6%±3.1%) and the proportion of PCNA stained follicles (26.4%±1.2%, P<0.05). In contrast, MEM+VC+EGF+FSH resulted the highest follicles diameter ((42.5±5.1) μm), follicles survival rate (59.7%±6.1%) and proportion of PCNA stained follicles (43.5%±4.1%, P<0.05). Ultrastructural analysis confirmed the integrity of follicles cultured in VC+EGF+FSH group, while follicles cultured in MEM+VC+EGF groups showed more degeneration characters. In conclusion, the addition of VC and EGF to culture medium inhibited follicular development, VC+EGF+FSH was the most effective treatment to maintain follicular integrity and promote sheep primordial follicular activation and growth during in vitro culture.