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Background: Glucose‐6‐phosphate dehydrogenase (G6PD) is an essential enzyme that protects red blood cells from oxidative damage. Although G6PD‐deficient alleles appear to confer a protective effect of malaria, the link with clinical protection against Plasmodium infection is conflicting. Methods: A case–control study was conducted on Bioko Island, Equatorial Guinea and further genotyping analysis used to detect natural selection of the G6PD A‐ allele. Results: Our results showed G6PD A‐ allele could significantly reduce the risk of Plasmodium falciparum infection in male individuals (adjusted odds ratio [AOR], 0.43; 95% confidence interval [CI], 0.20–0.93; p <.05) and homozygous female individuals (AOR, 0.11; 95% CI, 0.01–0.84; p <.05). Additionally, the parasite densities were significantly different in the individuals with different G6PD A‐ alleles and individual levels of G6PD enzyme activity. The pattern of linkage disequilibrium and results of the long‐range haplotype test revealed a strong selective signature in the region encompassing the G6PD A‐ allele over the past 6,250 years. The network of inferred haplotypes suggested a single origin of the G6PD A‐ allele in Africans. Conclusion: Our findings demonstrate that glucose‐6‐phosphate dehydrogenase (G6PD) A‐ allele could reduce the risk of P. falciparum infection in the African population and indicate that malaria has a recent positive selection on G6PD A‐ allele. [ABSTRACT FROM AUTHOR]