Tumor-associated macrophages (TAMs), which generally exhibit an M2-like phenotype, play a critical role in tumor development. Triptolide exerts a unique bioactive spectrum of anticancer activities. The aim of this study was to determine whether triptolide has any effect on the activation of TAMs and the production of tumor-promoting mediators. ICR-1 mice with azoxymethane/dextran sulfate sodium (AOM/DSS)-induced colon tumors and BALB/c mice co-inoculated with 4T1 cells and M2-polarized RAW264.7 cells were used to examine whether the inhibitory effect of triptolide on tumor progression was mediated by the targeting of TAMs. Real-time PCR, Western blot, immunofluorescence staining, and flow cytometry assays were performed to determine the expression of cell surface markers and cytokine production. The results showed that triptolide inhibited macrophage differentiation toward the M2 phenotype and abolished M2 macrophage-mediated tumor progression. Furthermore, triptolide inhibited the expression of M2 markers, such as CD206, Arginase 1, and CD204, and inhibited the secretion of anti-inflammatory cytokines. Thus our study indicated that triptolide selectively inhibited the functions of M2-polarized macrophages and TAMs, and this inhibitory effect of triptolide on TAM viability, differentiation, and cytokine production might elucidate the major mechanisms underlying its antitumor activity. Our findings provide important information for the potential clinical application of triptolide in cancer therapy.