An arabinogalactan 4‐β‐d‐galactanohydrolase was purified to a homogeneous state from the culture filtrate of a strain of Bacillus subtilis. The enzyme have a molecular mass of 36 kDa and an isoelectric point of pH 7.9. The enzyme is most active at around pH 6.5–7 and at 60°C, and is stable between pH 6–10 and below 55°C. Hg2+ and Cu2+ inhibit the activity. The enzyme hydrolyze soybean arabinogalactan which contains β‐1,4‐galactosidic linkages in its main chain structure, but not other polysaccharides with β‐1,3‐galactosidic linkages. The hydrolysis products from soybean arabinogalactan are predominantly galactobiose with a small amount of galactotetraose. The enzyme is an exo‐enzyme and the ability to transfer galactobiose to other galactobiose molecules is indicated by the formation of galactotetraose.