Neuropeptide S (NPS) is a naturally occurring appetite stimulant, associated with anxiety, stress, and excitement regulation. Neuropeptide S serves as a hypothalamic energy regulator that enhances food intake with a reduced level of satiety. NPS activates fat angiogenesis and the proliferation of new adipocytes in obesity. NPS has an established role in energy regulation by many pre-clinical investigations; however we have limited data available to support this notion in humans. We found significant association of Neuropeptide S receptor (NPSR1) Asn107Ile (rs324981, A>T) polymorphism with obese male participants. The current investigation carried out genotype screening of NPSR1 allele to assess the spectrum of the Asn107Ile polymorphism in obese and healthy Pakistani individuals. We revealed a significant (p = 0.04) difference between AA vs TT + AT genotype distribution of NPSR1 (SNP rs324981,) between obese and healthy individuals (p = 0.04). In this genotype analysis of (SNP rs324981) of the NPSR1 gene, T allele was marked as risk allele with higher frequency in the obese (38%) compared to its frequency in the controls (25%). Single Nucleotide Polymorphism (SNP, rs324981) Asn107Ile of NPSR1gene, that switches an amino acid from Asn to Ile, has been found associated with increased susceptibility to obesity in Pakistani individuals. Furthermore, molecular simulation studies predicted a lower binding affinity of NPSR1 Asn107Ile variant to NPS than the wild-type consistent with the genotype studies. These molecular simulation studies predict a possible molecular mechanism of this interaction by defining the key amino acid residues. However, a significantly (p<0.0001) lower concentration of NPS was recorded independent of genotype frequencies in obese subjects compared to healthy controls. We believe that large scale polymorphism data of population for important gene players including NPSR1 will be more useful to understand obesity and its associated risk factors.