Phagocyte NADPH oxidase activity is known to control parasite infection through reactive oxygen species production. We recently described functional/spatial interaction between NADPH oxidase and PDI in vascular smooth cells. We hypothesized that PDI supports phagocytosis during parasite infection. Here, we first confirmed the role of PDI in NADPH oxidase oxygen burst (Amplex-Red assay) triggered by PMA stimulation of macrophages J774 transfected with sense and antisense PDI cDNA. PDI over-expression markedly amplified oxygen burst (60-80% above control in 5-10 min), while PDI-antisense nearly completely inhibited it. A similar inhibition was obtained by knocking-down PDI using siRNA. PDI role during phagocytosis was assessed in in vitro infection of macrophages by promastigotes of Leishmania chagasi. Microscopy analysis showed that murine J774 or mouse peritoneal macrophages pre-incubated with PDI inhibitors DTNB (IC₅₀ 750mM), Bacitracin (IC₅₀ 500mM), p-phenylarsine oxide (PAO) (IC₅₀ 5mM) and neutralizing Ab-PDI (d=1/100) promoted significant decrease in Phagocytic Index (Pi). PDI mediation of parasite phagocytosis was further supported by gain and loss-of-function experiments in J774 macrophages transfected with sense or antisense PDI cDNA. Importantly, during phagocytosis, macrophage PDI translocates to membrane fraction in a way prevented by 5 mg/ml Brefeldin-A, a Golgi disrupting agent. Pi was closely dependent on redox state of promastigotes, as indicated by phagocytosis assays performed with macrophages J774 pre-incubated with PDI inhibitors and infected with parasites previously reduced (1mM DTT) or oxidized (1mM H₂O₂). In these conditions, Pi was 30% higher with the parasite in reduced state. In addition, assays in which we added exogenous PDI (100nM) to culture medium showed large Pi decrease in reducing (5mMGSH/0.5mMGSSG) vs. oxidizing (0.5mMGSH/5mMGSSG) conditions. We further searched for potential protein targets in the parasite for macrophage PDI. Using DTNB-PAO assay, dithiol protein content in parasite membrane fraction was estimated as 20%. Immunoprecipitation studies revealed that macrophage PDI closely interacts with an yet identified 100 KDa protein of Leishmania membrane fraction. Together, these data suggest that PDI not only sustains phagocyte NADPH oxidase activity, but it is also required for efficient phagocytosis in parasite-host interaction.