The vascular loops that traverse the melatonin-sensitive pars tuberails (PT)/infundibular border of the ovine pituitary gland undergo a seasonal change in morphology. Vascular remodelling is regulated by pro-angiogenic (VEGFxxx) and anti-angiogenic (VEGFxxxb) VEGF isoforms, which stimulate or reduce angiogenesis and vascular permeability, respectively. It is hypothesised that changes in the pituitary microvasculature in photoperiodic species are controlled by differential VEGF isoform production by the PT, regulated by melatonin signals throughout the annual reproductive cycle. In this study, primary PT cultures were generated from luteal phase sheep during the breeding season (BS, winter solstice), and anoestrous sheep in the non-BS (NBS, summer solstice). Cells remained in culture for 6 days and were treated daily with: i) media for 24h (control); ii) 1hr:23hr melatonin:media (acute winter); iii) 16hr:8hr melatonin:media (chronic winter); iv) 1hr:23hr melatonin:media (acute summer); and v) 8hr:16hr melatonin:media (chronic summer). Melatonin (10-6 Molar) treatments were started at the time of the endogenous melatonin nocturnal rise in the respective season. Media were collected at each time point and VEGF isoforms measured by ELISA. In the BS, acute melatonin did not affect VEGFxxxb secretion (p>0.05). In contrast, winter and summer chronic melatonin treatments caused a thirteen-fold (p<0.01) and four-fold (p<0.05) increase in VEGFxxxb, respectively. Critically, total VEGF (PanVEGF) levels were not affected by melatonin (p>0.05). In the NBS, chronic (p<0.01), but not acute (p>0.05), melatonin treatments stimulated VEGFxxxb output, with similar responses over the first 3 days. However, the response to the winter treatment increased thereafter (p<0.05), demonstrating that a continued BS pattern of melatonin, during the NBS, can revert VEGF production to a winter output. These results show that VEGF isoform expression in ovine PT is modified by melatonin duration, therefore providing a mechanism for regulation of pituitary vessel morphology and changes in fertility during the annual reproductive cycle.