CD40, a costimulatory molecule expressed on macrophages, induces expression of interleukin 12 (IL-12) in uninfected macrophages and IL-10 in macrophages infected with Leishmania major. IL-12 suppresses, whereas IL-10 enhances, L. major infection. The mechanisms that regulate this difference in CD40-induced cytokine production remain unclear, but it is known that L. major depletes cholesterol. Here we show that cholesterol influenced the assembly of distinct CD40 signalosomes. Depletion of membrane cholesterol inhibited the assembly of an IL-12-inducing CD40 signalosome containing the adaptors TRAF2, TRAF3 and TRAF5 and the kinase Lyn and promoted the assembly of an IL-10-inducing CD40 signalosome containing the adaptor TRAF6 and the kinase Syk. Thus, cholesterol depletion might represent an immune-evasion strategy used by L. major.