Differentiation-inducing factor-1 (DIF-1) identified in Dictyostelium discoideum has been reported to exhibit anti-tumor effects on several cancer cells, including breast cancer cells. Tumor progression is associated with not only cancer cells themselves but also the tumor microenvironment (TME). In particular, breast cancer tumor is dominated by stromal tissue of TME in which cancer-associated fibroblasts (CAF) are well known as one of the major components of TME. Here, we investigated whether DIF-1 could affect CAF in triple negative breast cancer (TNBC). In the in-vivo systems, we used a tumor-bearing mouse model in which 4T1-GFP TNBC cells were inoculated into mammary fat pad. DIF-1 was intragastrically administered 5 days a week and breast tumors were evaluated at Day 14. The immunohistochemistry showed that DIF-1 decreased the expression of a CAF‘s marker α-SMA. In the in-vitro systems, 4T1 cells and mouse primary dermal fibroblasts (DFBs) were used. C-X-C type chemokines (CXCL1, CXCL2 and CXCL5)/C-X-C type chemokine receptor 2 (CXCR2) axis acts as a communication mediator between cancer cells and CAF. In cultured 4T1 cells, DIF-1 inhibited CXCL1, CXCL2 and CXCR2 mRNA expressions. Co-culture with 4T1 cells induced an increase of CXCL1, CXCL2 and CXCL5 mRNA expressions in DFBs. Co-culture with DIF-1 treated 4T1 cells significantly suppressed the increase of CXCLs mRNA expressions. These results suggested that DIF-1 might downregulate CAF progression by inhibiting both CXCLs secretion in DFBs and CXCR2 expression in 4T1 cells.