Twain most clearly and famously criticizes imperialism in his 1901 essay "To the Person Sitting in Darkness". He criticizes the imperialistic activities of several countries, including Germany and Great Britain in the satirical essay, but since the question refers to American imperialism, this answer will focus on his American critique.
First, he claims that American behavior in the Philippines, which William McKinley and Congress had decided to annex after American victory in the Spanish-American War "liberated" it from American control, was contrary to American principles. Rather than annexing the islands, Twain contends, the United States should have liberated them, warning foreign powers not to meddle with them. But the United States did not do this, because they were tempted to play the "game" of imperialism that characterized relations among European powers. The result was a brutal war in the Philippines, one which raged on even as Twain was writing.
Second, Twain basically argues that imperialism is motivated by greed, not any desire, stated by missionaries and imperialists, to bring the blessings og "civilization" to people "sitting in darkness". Twain mocks these pretensions of what he calls the "Blessings of Civilization Trust" (a reference to the large monopolies that dominated the American economy at the time), arguing that in the end, imperialism is about exploiting poor, colonial peoples for the profit of rich nations.