Although characterised predominantly for its role in the brain, the neuropeptide kisspeptin has previously been shown to potentiate glucose-stimulated insulin through its receptor, GPR54; expressed abundantly in β-cells. We have previously reported a physiological role for kisspeptin signalling in the islet adaptation to pregnancy, using a β-cell specific GPR54 knockout mouse line (βGPR54ko). The aim of the present study was to examine the effects of impaired β-cell kisspeptin signalling on glucose homeostasis with aging in males and females using the βGPR54ko model. Male and female βGPR54ko and control (con) mice were fed chow (CD) or high-fat high-sugar diet (HFHSD) from 12 weeks of age. At 18 weeks, intraperitoneal glucose tolerance tests (IPGTT) and intraperitoneal insulin tolerance tests (IPITT) were carried out on all mice. IPGTT and IPITT were repeated 8 weeks later and every subsequent 4 weeks. At 18 weeks, HFHSD male and female mice had impaired glucose tolerance compared to CD (P<0.001), however, there was no significant difference between con and βGPR54ko mice (males: P=0.5, females: P=0.4). By 38 weeks, glucose tolerance in βGPR54ko HFHSD females was significantly impaired compared to HFHSD con mice (AUC con: 1919 vs. βGPR54ko: 3463; P=0.0017). This effect in βGPR54ko mice was not seen in HFHSD males (P=0.4). At subsequent time points HFHSD βGPR54ko females continued to exhibit impaired tolerance compared to equivalent controls (P=0.039), whilst no significant differences were observed in males (P=0.3). HFHSD significantly reduced insulin sensitivity in both males and females, as assessed by IPITT (week 38; males: P=0.03, females: P=0.006), but β-cell GPR54 knockout had no effect at any age (week 38; males: P=0.09, females: P=0.6). These results suggest that endogenous kisspeptin acting on the β-cells plays a physiological role in maintaining healthy glucose homeostasis with age in females, but that this role is less significant in males.