The incidence of cardiovascular disease, including inflammatory heart diseases like myocarditis, is increased in men. Similarly, male BALB/c mice infected with coxsackievirus B3 (CVB3) develop more severe acute inflammation in the heart compared to females. To better understand the effect of male sex hormones on cardiac inflammation, we gonadectomized (Gdx) male BALB/c mice and examined acute CVB3-induced myocarditis compared to sham controls. Viral replication in the heart was not significantly altered between Gdx and sham mice. However, gonadectomy significantly reduced testosterone levels and inflammation in the heart. FACS analysis of cell populations isolated from the heart revealed that CD11b(+) cells were significantly reduced in Gdx males. However, a GR1(+)F4/80(+) subset of CD11b(+) cells was significantly increased. Because this subset also expressed the interleukin (IL)-4R and IL-10, we refer to these cells as "alternatively activated" or M2 macrophages. A greater percentage of M2 macrophages in Gdx males expressed the inhibitory receptor Tim-3, while fewer expressed IL-1beta and IL-10. Only M2 macrophages upregulated TLR4 and Tim-3, whereas GR1(-)IL-4R(lo) macrophages did not. Additionally, IL-4(+)CD4(+) Th2 cells, Foxp3(+) regulatory T (Treg) cells and Tim-3(+)CD4(+) T cells were significantly increased in the heart following Gdx. Thus, we report for the first time that the inhibitory receptor Tim-3 is expressed on M2 macrophages. Our findings show that sex hormones and/or other mediators released from the testes inhibit anti-inflammatory populations in the heart including Tim-3(+) M2, Tim-3(+)CD4(+) T cells, Th2 and Treg resulting in more severe acute cardiac inflammation in males following CVB3 infection.