Neuropeptide Y (NPY) is one of the most potent orexigenic neuropeptides in mammals. Although NPY is widely expressed within the brain, the preponderance of research investigating NPY’s effects on appetite have focussed on the hypothalamus. In the present work, we examined the distribution, neurochemical phenotype and function of NPY cells within the key homeostatic brain region, the nucleus of the solitary tract (NTS) using NPY Humanized Renilla Green Fluorescent protein (NPY-GFP; n=38) and NPY-Cre mice (n=17). All animal studies were conducted in accordance with The Animals (Scientific Procedures) Act 1986. Using in situ hybridisation histochemistry (ISSH) to label endogenous Npy mRNA and immunohistochemistry to label GFP, we observed that nearly all NPY-GFP cells co-expressed Npy mRNA (n=6). These findings validate the NPY-GFP line. Next, we assessed the distribution and neurochemical phenotype of NPY neurons within the NTS. The majority of NPY neurons are localised between -7.20 and -7.56 from bregma (n=29). As expected, the majority (63.62±4.92%) of NPY-GFP cells co-expressed paired-like homeobox 2b (Phox2b), the transcription factor essential for the development of the autonomic nervous system (n=4), and none co-expressed the neurotransmitter acetylcholine (using choline acetyltransferase), which is localised outside the NTS (n=3).  Similar to the arcuate nucleus of the hypothalamus, NPY-GFP co-expression was observed with the neurotransmitter GABA (using glutamate decarboxylase67, GAD67; 22.20±2.78%; n=3). Perhaps counter-intuitively, a larger subset (54.66±7.50%; n=7) of NPY-GFP cells showed co-localisation with the anorectic protein nesfatin, although nesfatin’s function within the NTS has not been well described. Roughly a quarter (29.20±7.75%; n=7) showed co-expression for the catecholamines (using tyrosine hydroxylase) which are also classically anorectic. No NPY-GFP cells were co-expressed with the enzyme for nitric oxide, nitric oxide synthase (nNOS) (n=4). To examine the function of NTS NPY neurons, NPY-Cre mice were stereotaxically injected with the Cre-dependant designer receptor exclusively activated by designer drug (DREADD-Gq-mCherry or DREADD-Gi-mCherry) into the NTS (n=8/9 per group) under gas (2-3% isoflurane in O2) anaesthesia. We observed that activating NPY NTS neurons (DREADD-Gq-mCherry) with designer drug clozapine-N-oxide (CNO, i.p., 1 mg/kg/10 ml) significantly increased food intake whereas inhibiting NPY NTS neurons (DREADD-Gi-mCherry) significantly decreased food intake compared with saline treatment (two-tailed paired t-test or Wilcoxon sign-rank test, p<0.05). These data provide the first detailed characterisation of NPY neurons within the NTS and reveal that this population of neurons regulate appetite in mice.