The heterodimeric glycoprotein hormone, follicle stimulating hormone (FSH) and its receptor (FSHR) are vital for reproductive function, driving ovarian function via follicular recruitment, selection and development. FSH exists as two predominant glycoforms in females; partially glycosylated (FSH21) and fully glycosylated (FSH24), based on differing glycosylation patterns of the β subunit. The FSH glycoforms have different bioactivities, with FSH21 displaying a higher binding affinity for FSHR, shorter circulating half-life and increased potency of Gas-dependent signalling pathways, than FSH24. FSH21 and FSH24 are co-secreted, with the ratio of FSH21:FSH24 changing with age, with FSH21 predominant in women of reproductive prime, and FSH24 predominant in menopausal/post-menopausal women. Little is known about how the ovarian steroid hormone environment regulates FSH21 and FSH24 functions. The aim of this study was to determine the effects of testosterone on FSH/FSHR-dependent signal pathway activation. The cell model was the human granulosa cell line (KGN) cells, that endogenously express FSHR. KGN cells were pre-treated ±500 nM testosterone for 48 h, and subsequently stimulated for 15 min with either FSH21 or FSH24. In the absence of testosterone pre-treatment, FSH21 increased ERK-phosphorylation in a concentration-dependent manner. The response was biphasic, with 0.01 nM FSH21 resulting in a 2.0-fold increase in ERK phosphorylation, and maximum response of 2.7-fold increase with 1 nM FSH21, and decreasing thereafter. Surprisingly, pre-treatment with testosterone attenuated FSH21 dependent ERK phosphorylation. Suggesting possible pathway cross-talk to regulate FSHR signalling. In the absence of testosterone, FSH24 also stimulated ERK-phosphorylation, but was less potent than FSH21. Pre-treatment with testosterone ablated FSH24-dependent ERK phosphorylation. These data suggest that the endocrine microenvironment plays an important role in regulating FSH/FSHR signalling, with the ovarian functional consequences to be determined.