It features a rattlesnake above the warning, "DON'T TREAD ON ME". The rattle snake had become a traditional symbol of the American Colonies. The most obvious reason for this is that the rattlesnake was only found in the American Colonies and nowhere else in the world. The origin of the slogan (Don't Tread On Me) pertains to the snakes deadly strike and the idea that it is best generally when left alone.
The snake on the flag is also roughly arranged in the shape of the American Colonies with the tip of the tail being the Florida peninsula and the head being New England.