Background: Plasmodium falciparum causes the deadliest form of malaria in humans. Upon infection, the host’s infected red blood cells (iRBCs) are remodelled by exported parasite proteins to provide a niche for parasite development and maturation. Methods: Here we analysed the role of three PHISTb proteins Pf3D7_0532400, Pf3D7_1401600, and Pf3D7_1102500 by expressing recombinant proteins and evaluated antibody responses against these proteins using immune sera from malaria-exposed individuals from Kenya and The Gambia in Africa. Results: Children and adults from malaria-endemic regions recognized the three PHISTb proteins. Responses against PHISTb proteins varied with malaria transmission intensity in three different geographical sites in Kenya (Siaya and Takaungu) and The Gambia (Sukuta). Antibody responses against PHISTb antigens Pf3D7_1102500 and Pf3D7_1401600 were higher in Sukuta, a low transmission region in Gambia, compared to Siaya, a high transmission region in western Kenya, unlike Pf3D7_0532400. Anti-PHIST responses indicate negative correlation between antibody levels and malaria transmission intensity for Pf3D7_1102500 and Pf3D7_1401600. We report a correlation in antibody responses between schizont and gametocyte extract, but this is not statistically significant (cor=0.102, p=0.2851, CI=95%) and, Pf3D7_0532400 (cor=0.11, p=0.249, CI=95%) and Pf3D7_1401600 (cor=0.02, p=0.7968, CI=95%). We report a negative correlation in antibody responses between schizont and Pf3D7_1102500 (cor=-0.008, p=0.9348, CI=95%). There is a correlation between gametocyte extract and Pf3D7_1401600 (cor=-0.0402, p=0.6735, CI=95%), Pf3D7_1102500 (cor=0.0758, p=0.4271, CI=95%) and Pf3D7_0532400 (cor=0.155, p=0.1028, CI=95%). Acquisition of anti-PHIST antibodies correlates with exposure to malaria for Pf3D7_0532400 (p=0.009) but not Pf3D7_1102500 and Pf3D7_1401600 (p=0.507 and p=0.15, respectively, CI=95%). Children aged below 2 years had the lowest antibody levels which do not correlate with age differences. Conclusions: Collectively, these findings provide evidence of natural immunity against PHISTb antigens that varies with level of malaria exposure and underscore their potential as possible serological markers to P. falciparum infection aimed at contributing to malaria control through vaccine development.