The anterior pituitary releases six different hormones that control virtually all aspects of vertebrate physiology, yet the molecular mechanisms underlying their Ca(2+)-triggered release remain unknown. A subset of the synaptotagmin (syt) family of proteins serve as Ca(2+) sensors for exocytosis in neurons and neuroendocrine cells, and are thus likely to regulate pituitary hormone secretion. Here we show that numerous syt isoforms are highly expressed in the pituitary gland in a lobe, and sex-specific manner. We further investigated a Ca(2+)-activated isoform, syt-9, and found that it is expressed in a subpopulation of anterior pituitary cells, the gonadotropes. Follicle-stimulating hormone (FSH) and syt-9 are highly co-localized in female, but not male, mice. Loss of syt-9 results in diminished basal and stimulated FSH secretion only in females, resulting in alterations in the oestrus cycle. This work uncovers a new function for syt-9 and reveals a novel sex difference in reproductive hormone secretion.