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Calcite is a common mineral in various types of rocks and mineral deposits, and its oxygen isotopic compositions are widely used to reconstruct palaeotemperature and to trace rock genesis and ore‐forming processes. However, the availability of high‐quality matrix‐matched calcite reference materials for oxygen isotope microanalysis is limited. A new natural calcite specimen (NJUCal‐1) was analysed in this study to investigate its potential as a reference material for microbeam oxygen isotope measurement. Detailed textural and major elemental examinations reveal no internal zoning of ground grains. During six measurement sessions, a total of 463 spot analyses of oxygen isotope ratios were applied on ~ 200 randomly selected grains using secondary ion mass spectrometry, Nanjing. Taking all six sessions together, the spot‐to‐spot repeatability is 0.28‰ (2s), and the repeatability is better than 0.31‰ (2s) for individual sessions, indicating that the NJUCal‐1 calcite is quite homogeneous in its oxygen isotopic compositions and can be used as a new reference material for microbeam oxygen isotope measurement. The mean δ18OVPDB value determined by gas source isotope ratio mass spectrometry is −22.58 ± 0.16‰ (2s, n = 22), which is proposed as the recommended value for this potential reference material. [ABSTRACT FROM AUTHOR]