Background Triple negative breast cancer (TNBC) is a breast cancer (BC) subtype that is characterized by its strong invasion and a high risk of metastasis. However, the specific mechanisms underlying these phenotypes are unclear. TUFT1 plays an important role in BC and impacts the proliferation and survival of BC cells. Recent studies have shown that TUFT1 mediates intracellular lysosome localization and vesicle transport by regulating Rab GTPase, but the relevance of this activity in TNBC is unknown. Therefore, our aim was to systematically study the role of TUFT1 in the metastasis and chemoresistance of TNBC. Methods We measured TUFT1, Rab5-GTP, and Rac1-GTP expression levels in samples of human TNBC by immunohistochemistry (IHC) and conducted univariate and multivariate analyses. shRNA-mediated knockdown and overexpression, combined with transwell assays, co-immunoprecipitation, a nude mouse xenograft tumor model, and GTP activity assays were used for further mechanistic studies. Results TUFT1 expression was positively correlated with Rab5-GTP and Rac1-GTP in the TNBC samples, and co-expression of TUFT1 and Rab5-GTP predicted poor prognosis in TNBC patients who were treated with chemotherapy. Mechanism studies showed that TUFT1 could activate Rab5 by binding to p85 alpha, leading to activation of Rac1 through recruitment of Tiam1, and concurrent down-regulation of the NF-kappa B pathway and proapoptotic factors, ultimately promoting metastasis and chemoresistance in TNBC cells. Conclusions Our findings suggest that the TUFT1/Rab5/Rac1 pathway may be a potential target for the effective treatment of TNBC.