Hypoxia‑induced carbonic anhydrase IX (CAIX) is involved in intracellular and extracellular pH regulation, which is critical for tumor growth and metastasis. CAIX is overexpressed in breast cancer and is associated with the poor survival of patients after radiotherapy. Therefore, we evaluated the cellular and radiobiological effects of CAIX inhibition in human breast cancer cells. We used CA9 siRNA and the CA inhibitor (CAI) U104, respectively, to inhibit CAIX expression and activity in basal triple‑negative MDA‑MB‑231 and luminal MCF‑7 cells under hypoxic conditions. We investigated the effects of CAIX inhibition on CA9 mRNA and CAIX protein level, as well as on CAIX activity, intracellular pH, proliferation, apoptosis, clonogenic survival, migration, cell cycle distribution and radiosensitivity. CA9 siRNA and CAI U104 decreased CA9 mRNA and CAIX protein level in MDA‑MB‑231 and MCF‑7 cells. Furthermore, incubation with CAI U104 significantly decreased carbonic anhydrase activity and reduced the intracellular pH. Additionally, CA9 siRNA or U104 reduced clonogenic survival, migration and the number of cells in the G0/G1 phase, induced apoptosis and demonstrated additive or synergistic effects in combination with irradiation. In conclusion, combination of CAIX inhibition and irradiation is a promising treatment strategy against breast cancer with hypoxia‑induced CAIX expression.