Otto I, Prince of Anhalt-Aschersleben (d. 25 June 1304), was a German prince member of the House of Ascania and ruler of the Principality of Anhalt-Aschersleben.

He was the eldest son of Henry II, Prince of Anhalt-Aschersleben by his wife Matilda, daughter of Otto the Child, Duke of Brunswick-Lüneburg. Otto received his name after his maternal grandfather.

After the death of his father in 1266, Otto and his younger brother Henry inherited the principality of Anhalt-Aschersleben as co-rulers; but, because they are underage, their mother Matilda asumed the regency of Aschersleben until 1275, were both brothers were declared adults.

Otto continued his co-rule with his brother without divided their lands until 1283, when Henry renounced to his rights and became a priest. Since then, Otto ruled alone until his own death.

In 1283 Otto married with Hedwig (b. ca. 1256 - d. aft. 14 December 1300), daughter of Henry III the White, Duke of Breslau and widow of Henry, eldest son of Albert II, Margrave of Meissen. They had three children:

1. Otto II, Prince of Anhalt-Aschersleben (d. 1315).
2. Sophie. married bef. 1308 to Count Ulrich III of Regenstein-Heimburg.
3. Elisabeth, married ca. 1300 to Count Frederick of Beichlingen-Rotenburg.