Follicle-stimulating hormone (FSH) exists as different major glycoforms that differ in glycosylation of the hormone-specific β-subunit. Tetra-glycosylated FSH (FSH²⁴) and tri-glycosylated FSH (FSH²¹) are the most abundant forms found in humans. Employing distinct readouts in HEK293 cells stably expressing the human FSH receptor, we here compared intracellular signaling triggered by human pituitary FSH preparations (FSH²¹and FSH²⁴) as well as by equine FSH (eFSH), and human recombinant FSH (recFSH), each exhibiting distinct glycosylation patterns. The potencies in eliciting cAMP production were eFSH>FSH²¹>FSH²⁴>recFSH, whereas in the ERK1/2 activation readout potencies were FSH²¹≥eFSH>recFSH>FSH²⁴. In β-arrestin 1/2 CRISPR/Cas9 HEK293 KO cells, FSH²¹ exhibited a preference towards β-arrestin-mediated ERK1/2 activation as revealed by a drastic decrease in pERK during the first 15min exposure to this glycoform. Further, exposure of β-arrestin1/2 KO cells to H89 additionally decreased pERK1/2 albeit to a significantly lower extent in response to FSH²¹. Concurrent silencing of both β-arrestin and PKA signaling, did not completely suppress pERK in response to FSH glycoform, suggesting that pathways other than those dependent on Gs-protein and β-arrestins also contribute to FSH-stimulated pERK1/2. All four FSH glycoforms stimulated iCa²⁺ accumulation through influx from Ca²⁺ channels as well as release from intracellular stores; however, iCa²⁺ accumulation in response to FSH²¹ was more dependent on the latter, suggesting differences in the mechanisms through which FSH glycoforms promote  iCa²⁺ accumulation.These data indicate that FSH glycosylation plays an important role in defining both the intensity of signal activation and the balance among mechanistically different pathways mediating activation of distinct signaling cascades.