Recent studies underscore the importance of crosstalk between tumor-associated macrophages (TAMs) and tumor cells in cancer progression and metastasis. In our study, AdCD68STAT3β, a recombinant adenovirus containing a STAT3β gene driven by CD68 macrophage-specific promoter, was used to suppress STAT3 and the downstream signaling pathways in TAMs. The results showed that STAT3β gene under the control of CD68 macrophage-specific promoter was only expressed in macrophages, which significantly inhibited the motility and invasion of breast cancer cells when co-cultured with 4T1 cells. Moreover, cell-specific STAT3β expression in TAMs extended survival of tumor-bearing mice and suppressed breast tumor growth, angiogenesis and metastasis, by regulating the crosstalk between tumor cells and TAMs. Therefore, our study provided a novel strategy for the antitumor effects of STAT3β.