Calreticulin (CALR) is a multifunctional protein that participates in various cellular processes, which include calcium homeostasis, cell adhesion, protein folding, and cancer progression. However, the role of CALR in breast cancer (BC) is unclear. Here, we report that CALR is overexpressed in BC compared with normal tissue, and its expression is correlated with patient mortality and stemness indices. CALR expression was increased in mammosphere cultures, CD24⁻CD44⁺ cells, and aldehyde dehydrogenase–expressing cells, which are enriched for breast cancer stem cells (BCSCs). Additionally, CALR knockdown led to BCSC depletion, which impaired tumor initiation and metastasis and enhanced chemosensitivity in vivo. Chromatin immunoprecipitation and reporter assays revealed that hypoxia-inducible factor 1 (HIF-1) directly activated CALR transcription in hypoxic BC cells. CALR expression was correlated with Wnt/β-catenin pathway activation, and an activator of Wnt/β-catenin signaling abrogated the inhibitory effect of CALR knockdown on mammosphere formation. Taken together, our results demonstrate that CALR facilitates BC progression by promoting the BCSC phenotype through Wnt/β-catenin signaling in an HIF-1–dependent manner and suggest that CALR may represent a target for BC therapy.