Following another period of decommissioning, in 1836 where she underwent extensive remodeling, Vincennes was fitted out as flagship for Lieutenant Charles Wilkes' U.S. South Sea Surveying and Exploring Expedition to the Antarctic region. The expedition sailed from Hampton Roads, Virginia in August 1838 and made exploratory surveys along the South American coast before making a preliminary survey of Antarctica in early 1839. Entering into the South Pacific in August and September 1839, cartographers aboard Vincennes drafted charts that are still useful today.

In Late November, Vincennes set sail once more for Antarctica and on January 30, 1840, land was sighted for the first time in the Antarctic region. Wilkes reported that he "saw the land gradually rising from the ice to a height of three thousand feet." Now that all were convinced of its existence, gave the land the name of the Antarctic Continent. The coast along which the American vessel sailed is today known as Wilkesland, a name given on German maps as early as 1841.

After additional south seas surveys and an extensive survey of Puget Sound in mid-1841, Vincennes dropped anchor in San Francisco, California on August 14. On November 1st, the sloop once again set a course for the far east, arriving in Manila Bay in early January 1842. From there, Vincennes set sail on the long westward leg home, arriving in Sandy Hook, New Jersey on June 10, 1842, almost four years after the beginning of the expedition (completing her third trip around the world).

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