Follicle stimulating hormone (FSH) is a key endocrine regulator of ovarian function. FSH is secreted as two macro-glycosylation variants; partially glycosylated FSH (FSH21/18) and fully glycosylated FSH (FSH24). FSH21/18 is more potent than FSH24 at binding to and activating FSHR. The ratio of FSH21/18:FSH24 has been shown to change with age, with FSH21/18 predominant at reproductive prime, and FSH24 predominant during peri-menopause/menopause. How these FSH glycosylation variants modulate ovarian follicle functions remains largely unknown. The aim of this study was to investigate the effect of FSH glycosylation variants of pre-antral follicle function. Pre-antral follicles were isolated from 3-5wk-old-C57BL/6 mice and treated ±10 ng/ml FSH21/18, FSH24, a ratio of 80:20 FSH21/18:FSH24 (to mimic reproductive prime), 50:50 FSH21/18:FSH24 (peri-menopause) or 20:80 FSH21/18:FSH24 (menopause) for up to 96 hrs. FSH21/18 and 80:20 FSH21/18:FSH24 increased follicle growth, in comparison to control, contrasting with FSH24 and 20:80 FSH21/18:FSH24. Survival rates were decreased in follicles treated with FSH24 or 20:80 FSH21/18:FSH24, with follicles undergoing basement membrane rupture and oocyte extrusion, increased Caspase3 gene and protein expression, and decreased markers of cell proliferation in FSH24 or 20:80 FSH21/18:FSH24 treated-follicles. Moreover, this additionally correlated with differential regulation of key genes modulating follicular functions. Pharmacological inhibitors of key FSH signal pathways suggests FSH21/18 and FSH24 initiate different FSHR signal pathway activation, which may determine their differential effects on follicle growth and survival. These data suggest that the nature of FSH glycosylation modulates follicular cellular environment to regulate follicle growth and survival and may underpin the increasing ovarian resistance to FSH observed during ageing.