Chemoresistance is a crucial barrier to limit the therapeutic outcome of breast cancer (BC), and the mechanism underlying chemoresistance development in BC is not fully understood. In this study, we aimed to investigate the potential involvement of miR-26b-5p/serglycin (SRGN) axis in BC drug resistance. The expression level of SRGN in drug-resistant BC cells was investigated by western blotting analysis, real-time quantitative PCR (qRT-PCR), immunohistochemical staining, and ELISA. Its expression between chemoresistant and sensitive patient samples was compared by qRT-PCR. Bioinformatics tool and dual-luciferase reporter assay were employed to identify miR-26b-5p as a regulator of SRGN. Functional assays were performed to examine cell proliferation, cell viability, apoptosis, migration, and invasion ability in vitro. Xenograft tumorigenesis experiment was conducted to evaluate the tumor suppressor effect of miR-26b-5p on chemoresistant BC cells. SRGN expression was significantly upregulated in both chemoresistant BC cell lines and chemoresistant patient samples. miR-26b-5p was identified as an upstream regulator of SRGN. Overexpression of miR-26b-5p downregulated SRGN expression, overcame chemoresistance, and suppressed cell proliferation, migration, and invasion in BC cells. Overexpression of miR-26b-5p also suppressed the tumorigenesis of chemoresistant BC cells in vivo. Mechanistically, the downregulation of SRGN by miR-26b-5p decreased the expression of breast cancer drug-resistant protein and multidrug-resistant protein 1 in chemoresistant BC cells. Our study identified miR-26b-5p as a tumor suppressor which targets SRGN to sensitize BC cells to chemotherapeutics. These results suggest that miR-26b-5p and SRGN may serve as potential biomarkers and targets for BC chemotherapy.