In seasonally breeding animals, the circadian and photoperiodic regulation of neuroendocrine system is important for precisely-timed reproduction. Kisspeptin, encoded by the Kiss1 gene, acts as a principal positive regulator of the reproductive axis by stimulating gonadotrophin-releasing hormone (GnRH) neurone activity in vertebrates. However, the precise mechanisms underlying the cyclic regulation of the kisspeptin neuroendocrine system remain largely unknown. The grass puffer, Takifugu niphobles, exhibits a unique spawning rhythm: spawning occurs 1.5-2 h before high tide on the day of spring tide every 2 weeks, and the spawning rhythm is connected to circadian and lunar-/tide-related clock mechanisms. The grass puffer has only one kisspeptin gene (kiss2), which is expressed in a single neural population in the preoptic area (POA), and has one kisspeptin receptor gene (kiss2r), which is expressed in the POA and the nucleus dorsomedialis thalami. Both kiss2 and kiss2r show diurnal variations in expression levels, with a peak at Zeitgeber time (ZT) 6 (middle of day time) under the light/dark conditions. They also show circadian expression with a peak at circadian time 15 (beginning of subjective night-time) under constant darkness. The synchronous and diurnal oscillations of kiss2 and kiss2r expression suggest that the action of Kiss2 in the diencephalon is highly dependent on time. Moreover, midbrain GnRH2 gene (gnrh2) but not GnRH1 or GnRH3 genes show a unique semidiurnal oscillation with two peaks at ZT6 and ZT18 within a day. The cyclic expression of kiss2, kiss2r and gnrh2 may be important in the control of the precisely-timed diurnal and semilunar spawning rhythm of the grass puffer, possibly through the circadian clock and melatonin, which may transmit the photoperiodic information of daylight and moonlight to the reproductive neuroendocrine centre in the hypothalamus.