Background: Rehmanniae radix preparata (RRP) has been used as a traditional medicine and is one of the most important oriental herbal medicines. However, the physical characteristics of RRP are not suitable for use in industry. The present study was undertaken to determined the preparation method of RRP powder and the physicochemical characteristics of RRP powder by milling under different pre-freezing temperatures. Methods and Results: Moisture content, powder yield, particle size, bulk density, compressive stress, extraction yield, and 5-HMF content of PRR powders by milling with pre-freezing temperatures (−20, −40, −60, and −80℃) were analyzed, and correlation among these factors was determined. Powder yield increased and particle size decreased in a pre-freezing temperature-dependent manner from −20 to −60℃. The Hausner ratio increased from 1.186 to 1.225 with decreasing temperature from −20 to −80℃, whereas compressive stress showed the opposite trend. Extraction yield and 5-HMF content were not significantly different between RRP powder. Significant correlations were observed among pre-freezing temperature and physical characteristics (e.g., yield, particle size, Hausner ratio, and compressive stress). Conclusions: These results suggest that the pre-freezing temperature is an important factor affecting the physical characteristics of PRR powder and applicable to the industrial production of RRP powder