The motto, "Don't tread on me" was found on flags flown by anti British colonists in the run-up to the American Revolution, usually accompanied by a picture of a rattlesnake.

The meaning was clear.

Benjamin Franklin, one of the first promoters of the rattlesnake image for the American colonies put it thus:

"“… (The rattlesnake) has no eye-lids; she may therefore be esteemed an emblem of vigilance. She never begins an attack, nor, when once engaged, ever surrenders…she is therefore an emblem of magnanimity and true courage. The weapons with which nature has furnished her, she conceals in the roof of her mouth, so that, to those who are unacquainted with her, she appears to be a most defenseless animal…but (these weapons) and their (resulting) wounds however small, are decisive and fatal: Conscious of this, she never wounds till she has generously given notice, even to her enemy, and cautioned him against the danger of treading on her. Was I wrong in thinking this a strong picture of the temper and conduct of America?”

The rattlesnake also appealed to Franklin as an image for the 13 American colonies because the snake was said to have 13 rattles on its tail.

Source:

http://goldstandard.hubpages.com/hub/Dont-Tread-On-Me-Flag-The-Significance-of-the-Rattlesnake