Tumor-associated macrophages (TAMs) have been implicated in promoting tumor progression and invasion. The onset and maintenance of tumor angiogenesis and lymphangiogenesis also seem to be partly driven by a group of polarized alternatively activated macrophages (aaMphi) in lung adenocarcinoma. Here, the aaMphi and classically activated macrophages (caMphi) were obtained using RAW264.7 cells via IL-4 and IFN-γ + LPS treatment, respectively. Co-inoculation of aaMphi with Lewis lung carcinoma (LLC) cells promoted tumor growth, increased lymph node metastasis, and reduced the survival in C57BL/6 mice bearing LLC. Furthermore, the effects of the activated macrophages on the lymphangiogenesis-related properties of lymphatic endothelial cells (LECs) were investigated in vitro. When LECs were cultured in macrophages conditioned medium or in a co-culture system of macrophages and LECs, aaMphi significantly promoted proliferation, migration, and tube-like formation of LECs. We identified high VEGF-C expression in aaMphi and low expression in caMphi as well as unactivated macrophages by ELISA and Western blotting. In LECs, co-culture with aaMphi resulted in a significant increase of mRNA levels of specific lymphatic marker VEGF receptor-3 and the homeobox gene Prox-1, as well as lymphangiogenic factor VEGF-C rather than VEGF-D by quantitative RT-PCR. Furthermore, enhanced LECs migration and capillary formation by co-culture with aaMphi were significantly inhibited by rVEGF receptor-3/Fc chimera. In conclusion, these data show that aaMphi play a critical role in tumor-induced lymphangiogenesis through up-regulating VEGF-C and increasing lymphangiogenesis-related behavior of LECs, which may contribute to lymphatic invasion in lung adenocarcinoma. J. Cell. Biochem. 107: 134–143, 2009. © 2009 Wiley-Liss, Inc.