Macrophages are a dominant leukocyte population in the tumor microenvironment and actively promote cancer progression. However, the molecular mechanism underlying the role of macrophages remains poorly understood. Here we show that polarized M2 macrophages enhance 3-phosphoinositide-dependent protein kinase 1 (PDPK1)-mediated phosphoglycerate kinase 1 (PGK1) threonine (T) 243 phosphorylation in tumor cells by secreting inter-leukin-6 (IL-6).This phosphorylation facilitates a PGK1-catalyzed reaction toward glycolysis by altering substrate affinity. Inhibition of PGK1 T243 phosphorylation or PDPK1 in tumor cells or neutralization of macrophage-derived IL-6 abrogates macrophage-promoted glycolysis, proliferation, and tumorigenesis. In addition, PGK1 T243 phosphorylation correlates with PDPK1 activation, IL-6 expression, and macrophage infiltration in human glioblastoma multiforme (GBM). Moreover, PGK1 T243 phosphorylation also correlates with malignance and prognosis of human GBM. Our findings demonstrate a novel mechanism of macrophage-promoted tumor growth by regulating tumor cell metabolism, implicating the therapeutic potential to disrupt the connection between macrophages and tumor cells by inhibiting PGK1 phosphorylation.