Abstract Triple-negative breast cancers (TNBC) with higher cancer stem-like populations are reported to exhibit a more aggressive metastatic phenotype. In the present study, we investigated the effects of flubendazole, a candidate drug for repurposing and its novel mechanism of action with a focus on targeting STAT3 signaling and breast cancer stem-like traits in TNBC. The effect of FLU on TNBC cell lines in vitro was evaluated in terms of cell viability, breast cancer stem cells (BCSC)-like properties and expression of STAT3 signaling-related factors. An orthotopic injection model with BCSC-enriched population was used to examine the effect of flubendazole on tumor growth and metastasis in vivo. Our results showed that the BCSC-enriched populations harbored a higher Aldefluor-positive population and markedly elevated levels of CD49f and ALDH1A1 as well as higher STAT3 activation in vitro. Flubendazole attenuated STAT3 activation, as demonstrated by a significant reduction in phospho-STAT3 levels as well as subsequent downregulation of its downstream effector cyclin D1. Sphere-forming ability was significantly suppressed following two pharmacological STAT3 inhibitor, LLL12 or S3I-201 challenge. Treatment with interleukin-6 (IL-6) was observed to significantly increase the number and volume of mammospheres, while flubendazole abolished this effect. Bioluminescence in vivo imaging (BLI) revealed that the BCSC-enriched populations exhibited enhanced metastasis with higher STAT3 activation, while flubendazole administration inhibited tumor growth and lung and liver metastasis, coinciding with decreased MMP-2 and MMP-9 levels in circulating blood. To our knowledge, these findings are the first to report that flubendazole reduces BCSC-enriched tumor burden and metastasis via STAT3 inactivation, implying that flubendazole treatment may have application in addressing metastasis. Citation Format: Eunhye Oh, Yoon-Jae Kim, Seojin Jang, Tae-Min Cho, Soeun Park, Jung Min Park, Minsu Park, Ji Young Kim, Jae Hong Seo. Flubendazole targets STAT3 signaling and distant metasis in triple-negative breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3457.