Metabolic stress resulting from either lack or excess of nutrients often causes infertility in both sexes. Kisspeptin-neurokinin B-dynorphin A (KNDy) neurons in the arcuate nucleus (ARC) has been suggested to be a key players in reproduction via direct stimulation of the pulsatile gonadotropin-releasing hormone (GnRH) and subsequent gonadotropin release in mammalian species. In this study, we investigated the effect of high-fat diet (HFD) on hypothalamic KNDy gene expression to examine the pathogenic mechanism underlying obesity-induced infertility in male and female rats. Male and female rats at 7 weeks of age were fed with either a standard or HFD for 4 months. In the male rats, the HFD caused a significant suppression of ARC Kiss1 and Pdyn gene expressions, but did not affect the plasma luteinizing hormone (LH) levels and sizes of the morphology of the testis and epididymis. In the female rats, 58% of the HFD-fed female rats exhibited irregular estrous cycles, whereas the remaining rats showed regular cycles. Two of the 10 rats that showed HFD-induced irregular estrous cycles showed profound suppression of LH pulse frequency and the number of ARC Kiss1-expressing cells, whereas the other females showed normal LH pulses and ARC Kiss1 expression. Our finding shows that suppression of ARC Kiss1 expression might be the initial pathological change of hypogonadotropic hypogonadism in HFD-fed male rats, while the obese-related infertility in the female rats may be mainly induced by KNDy-independent pathways. Taken together, ARC kisspeptin neurons in male rats may be susceptible to HFD-induced obesity compared with those in female rats.