<p>Continuous exposure to <i>Plasmodium falciparum</i> (Pf) has been associated with alterations in B cells. We investigated the effect of controlled human malaria infection (CHMI) on B cell phenotypes in individuals with different Pf immunity status: malaria-naïve, immunized with PfSPZ-CVac and semi-immune (lifelong-exposed) volunteers. Compared to naïve, semi-immune but not vaccinated individuals, had increased baseline frequencies of immature B cells (CD19⁺CD10⁺), active naive (IgD⁺CD27⁻CD21⁻) B cells, active atypical (IgD⁻CD27⁻CD21⁻) memory B cells (MBCs), active classical (IgD⁻CD27⁺CD21⁻) MBCs and CD1c⁺-B cells but lower frequencies of some IgG⁺-B cells. The frequencies of CD1c⁺ active atypical MBCs correlated positively with anti-Pf antibodies and negatively with circulating eotaxin levels, while the opposite was observed for IgG⁺ resting atypical MBCs. During early blood-stage infection (day 11 after CHMI), there was an expansion of resting classical (IgD⁻CD27⁺CD21⁺) MBCs in all three groups. Vaccination, compared to placebo, altered the effect of CHMI on B cells, showing a positive association with resting classical MBCs (β = 0.190, 95% CI 0.011–0.368) and active naïve-PD1⁺ (β = 0.637, 95% CI 0.058 to 1.217) frequencies, and a negative one with CD1c⁺ resting atypical MBCs (β = − 0.328, 95% CI − 0.621 to − 0.032). In addition, the sickle cell trait in semi-immune subjects altered the effect of CHMI on several B cells. In conclusion, lifelong but not vaccine exposure to malaria was associated with increased frequencies of multiple B cell subsets, with higher and lower percentages of CD1c and IgG expressing-cells, respectively. A single infection (CHMI) induces changes in B cell frequencies and is modulated by sickle cell trait and malaria-immunity status.</p><p><i>Clinical Trials Registration</i> NCT01624961, NCT02115516, and NCT02237586.</p>