•High-accuracy mass calibration of sub-milligram weights was realized by using a recently developed automatic weight transfer apparatus and an analytical calibration method that is often used for mass calibration of small-mass weights.•The mass calibrations of sub-milligram weights were realized with standard uncertainties smaller than those of previous studies performed by other NMIs.•To improve the calibration uncertainty of the sub-milligram weights achieved in this study, it is necessary to improve the uncertainty of calibrated masses of reference weights, update the balance used, and stabilize the measurement environment. Mass standards are essential for the precise measurement of mass. Due to the increasing demand for small mass measurements, it is essential to be able to disseminate the sub-milligram mass standard from the National Metrology Institutes. Recently, the National Metrology Institute of Japan has developed an apparatus for mass calibration of sub-milligram weights by automatically transporting the weights. Consequently, the masses of the weights can be calibrated accurately and reliably. Sub-multiple calibration is often used for accurate mass calibration of weights smaller than 1 kg. In this study, by combining this method with the developed apparatus, the mass calibration uncertainty of sub-milligram weights was improved. The mass calibrations of 0.1 mg, 0.2 mg, and 0.5 mg weights were performed with standard uncertainties of 0.019 µg, 0.023 µg, and 0.040 µg, respectively. These uncertainties were smaller than the calibration uncertainties obtained by manually transporting sub-milligram weights to an ultra-micro balance.