The aim of our study was to investigate the role of S180L polymorphism in modulation of acquisition of malaria caused by Plasmodium falciparum in a small group of Pakistani population. A total of 133 individuals including 60 controls and 73 patients of malaria, caused by Plasmodium falciparum, were genotyped using allele-specific PCR. Ninety-two samples successfully demonstrated the PCR amplification results, while forty-one samples could not be genotyped due to failure in PCR amplification. The allele frequency for S180L polymorphism was deviant from Hardy-Weinberg equilibrium (HWE) of the population under observation. Association was found between the observed polymorphism and the occurrence of malaria caused by Plasmodium falciparum (p = 0.01). Chances of malaria caused by Plasmodium falciparum were low in CC genotype carriers in comparison to other genotypes (Odds ratio: 0.3016; 95% CI: 0.124-0.729). The present findings suggest that S180L polymorphism is important in modulating the probability of acquisition of malaria caused by Plasmodium falciparum in Pakistani population. The CC genotype plays a protective role in local population against this type of malaria.