The self-renewal and differentiation of hematopoietic stem cells (HSCs) in bone marrow are essential to replenish all blood cell types, but how this process is influenced by diet remains largely unclear. Here we show that a diet rich in fish oils promotes self-renewal of HSCs and extramedullary hematopoiesis. Chronic intake of a fish oil-rich diet increases the abundance of HSCs, alters the hematopoietic microenvironment, and, intriguingly, induces the expression of matrix metalloproteinase 12 (MMP12) in the bone marrow. Pointing to a direct effect of fish oil on MMP12 expression, omega-3 polyunsaturated fatty acids induce the expression of MMP12 in a dose-dependent manner in bone marrow cells. Importantly, down-regulation of MMP12 activity using an MMP12-specific inhibitor attenuates diet-induced myelopoiesis in both bone marrow and spleen. Thus, a fish oil-rich diet promotes hematopoiesis in the bone marrow and spleen, in part via the activity of MMP12. Taken together, these data provide new insights into diet-mediated regulation of hematopoiesis.