BackgroundS100A6 and murine double minute 2 (MDM2) are important cancer-related molecules. A previous study identified an interaction between S100A6 and MDM2 by size exclusion chromatography and surface plasmon resonance experiments. The present study investigated whether S100A6 could bind to MDM2 in vivo and further explored its functional implication.MethodsCo-immunoprecipitation, glutathione-S-transferase pull-down assay, and immunofluorescence were performed to determine the in vivo interaction between S100A6 and MDM2. Cycloheximide pulse-chase assay and ubiquitination assay were performed to clarify the mechanism by which S100A6 downregulated MDM2. In addition, clonogenic assay, WST-1 assay, and flow cytometry of apoptosis and the cell cycle were performed and a xenograft model was established to evaluate the effects of the S100A6/MDM2 interaction on growth and paclitaxel-induced chemosensitivity of breast cancer. The expressions of S100A6 and MDM2 in patients with invasive breast cancer were analyzed by immunohistochemistry. In addition, the correlation between the expression of S100A6 and the response to neoadjuvant chemotherapy was statistically analyzed.ResultsS100A6 promoted the MDM2 translocation from nucleus to cytoplasm, in which the S100A6 bound to the binding site of the herpesvirus-associated ubiquitin-specific protease (HAUSP) in MDM2, disrupted the MDM2–HAUSP–DAXX interactions, and induced the MDM2 self-ubiquitination and degradation. Furthermore, the S100A6-mediated MDM2 degradation suppressed the growth of breast cancer and enhanced its sensitivity to paclitaxel both in vitro and in vivo. For patients with invasive breast cancer who received epirubicin and cyclophosphamide followed by docetaxel (EC-T), expressions of S100A6 and MDM2 were negatively correlated, and high expression of S100A6 suggested a higher rate of pathologic complete response (pCR). Univariate and multivariate analyses showed that the high expression of S100A6 was an independent predictor of pCR.ConclusionThese results reveal a novel function for S100A6 in downregulating MDM2, which directly enhances sensitivity to chemotherapy.Supplementary InformationThe online version contains supplementary material available at 10.1186/s13058-023-01657-w.