AbstractSplenomegaly frequently occurs in patients with Plasmodium falciparum (Pf) or P. vivax (Pv) malarial anemia, but mechanisms underlying this co‐occurrence are unclear. In malaria‐endemic Papua, Indonesia, we prospectively analyzed red blood cell (RBC) concentrations in the spleen and spleen‐mimetic retention in 37 subjects splenectomized for trauma or hyperreactive splenomegaly, most of whom were infected with Plasmodium. Splenomegaly (median 357 g [range: 80–1918 g]) was correlated positively with the proportion of red‐pulp on histological sections (median 88.1% [range: 74%–99.4%]; r = .59, p = .0003) and correlated negatively with the proportion of white‐pulp (median 8.3% [range: 0.4%–22.9%]; r = −.50, p = .002). The number of RBC per microscopic field (>95% uninfected) was correlated positively with spleen weight in both Pf‐infected (r = .73; p = .017) and Pv‐infected spleens (r = .94; p = .006). The median estimated proportion of total‐body RBCs retained in Pf‐infected spleens was 8.2% (range: 1.0%–33.6%), significantly higher than in Pv‐infected (2.6% [range: 0.6%–23.8%]; p = .015) and PCR‐negative subjects (2.5% [range: 1.0%–3.3%]; p = .006). Retained RBCs accounted for over half of circulating RBC loss seen in Pf infections. The proportion of total‐body RBC retained in Pf‐ and Pv‐infected spleens correlated negatively with hemoglobin concentrations (r = −.56, p = .0003), hematocrit (r = −.58, p = .0002), and circulating RBC counts (r = −.56, p = .0003). Splenic CD71‐positive reticulocyte concentrations correlated with spleen weight in Pf (r = 1.0; p = .003). Retention rates of peripheral and splenic RBCs were correlated negatively with circulating RBC counts (r = −.69, p = .07 and r = −.83, p = .008, respectively). In conclusion, retention of mostly uninfected RBC in the spleen, leading to marked congestion of the red‐pulp, was associated with splenomegaly and is the major mechanism of anemia in subjects infected with Plasmodium, particularly Pf.