Nesfatin-1 is an 82 amino acid N-terminal fragment of nucleobindin2 that was consistently shown to reduce dark phase food intake upon brain injection in rodents. We recently reported that nesfatin-1 1–82 injected intracerebroventricularly (icv) reduces dark phase feeding in mice. Moreover, intraperitoneal injection of mid-fragment nesfatin-1 (nesfatin-1 30–59) mimics the food intake-reducing effects of nesfatin-1 1–82, whereas N-terminal (nesfatin-1 1–29) and C-terminal fragments (nesfatin-1 60–82) did not. We therefore characterized the structure-activity relationship of nesfatin-1 injected icv to influence the dark phase meal pattern in mice. Mouse nesfatin-1 1–29, nesfatin-1 30–59, nesfatin-1 60–82 or vehicle was injected icv in freely fed C57Bl/6 mice immediately before the dark phase and food intake was monitored using an automated episodic feeding monitoring system. Nesfatin-1 30–59 (0.1, 0.3, 0.9 nmol/mouse) induced a dose-related reduction of 4-h food intake by 28%, 49% and 49% respectively resulting in a 23% decreased cumulative 24-h food intake compared to vehicle ( p<0.05). The peak reduction occurred during the 3 rd (−96%) and 4 th hour (−91%) post injection and was associated with a reduced meal frequency (0–4h: −47%) and prolonged inter-meal intervals (3.1-times) compared to vehicle ( p<0.05), whereas meal size was not altered. In contrast, neither nesfatin-1 1–29 nor nesfatin-1 60–82 reduced dark phase food intake at equimolar doses although nesfatin-1 60–82 prolonged inter-meal intervals (1.7-times, p<0.05). Nesfatin-1 30–59 is the active core of nesfatin-1 1–82 to induce satiety indicated by a reduced meal number during the first 4h post injection. The delayed onset may be indicative of time required to modulate other hypothalamic and medullary networks regulating nocturnal feeding as established for nesfatin-1.