Age-related LH surge dysfunction in middle-aged rats is characterized, in part, by reduced responsiveness to estradiol (E2)-positive feedback and reduced hypothalamic kisspeptin neurotransmission. Vasoactive intestinal peptide (VIP) neurons in the suprachiasmatic nucleus project to hypothalamic regions that house kisspeptin neurons. Additionally, middle-age females express less VIP mRNA in the suprachiasmatic nucleus on the day of the LH surge and intracerebroventricular (icv) VIP infusion restores LH surges. We tested the hypothesis that icv infusion of VIP modulates the LH surge through effects on the kisspeptin and RFamide-related peptide-3 (RFRP-3; an estradiol-regulated inhibitor of GnRH neurons) neurotransmitter systems. Brains were collected for in situ hybridization analyses from ovariectomized and ovarian hormone-primed young and middle-aged females infused with VIP or saline. The percentage of GnRH and Kiss1 cells coexpressing cfos and total Kiss1 mRNA were reduced in saline-infused middle-aged compared with young females. In young females, VIP reduced the percentage of GnRH and Kiss1 cells coexpressing cfos, suggesting that increased VIP signaling in young females adversely affected the function of Kiss1 and GnRH neurons. In middle-aged females, VIP increased the percentage of GnRH but not Kiss1 neurons coexpressing cfos, suggesting VIP affects LH release in middle-aged females through kisspeptin-independent effects on GnRH neurons. Neither reproductive age nor VIP affected Rfrp cell number, Rfrp mRNA levels per cell, or coexpression of cfos in Rfrp cells. These data suggest that VIP differentially affects activation of GnRH and kisspeptin neurons of female rats in an age-dependent manner.