Here's a Google search for references to the Gadsden flag slogan on their site:
http://www.google.com/search?hl=en&safe=off&q=%22don%27t+tread+on+me%22+site%3Agopusa.com&btnG=Searchhttp://www.gopusa.com/forum/showthread.php?t=52857">This contributor (Post #3) used the slogan to express unhappiness that Barack Obama is popular around the world, calling it "a huge positive for McCain and Palin" and claiming that "Americans do not like to be told how to run our business by people from other countries."
http://www.gopusa.com/forum/showthread.php?t=59402">Here (Post #5), it's directed at Somali pirates. No problem with that, I guess.
http://www.gopusa.com/forum/showthread.php?t=59447&page=2">Here's one of many reports of uses of the slogan at "tea parties" (Post #17).
http://www.gopusa.com/forum/showthread.php?t=19219&page=21">Here (Post 205), it's somehow tied to a plea to save Terri Schiavo.
http://www.gopusa.com/forum/archive/index.php/t-34125.html">Here, it's used in agreement with a call for all-out American imperialism, with a fond reference to the Roman Empire.
And finally, check this out!
http://www.gopusa.com/theloft/wp-print.php?p=664">Here, it's used against John McCain!
You can't make a good deal with bad people (like McCain), no matter how lengthy the contract might be. Politicians, particularly those who have hijacked the Republican Party, are dishonorable people. We might as well vote for Democrats, because that is what we will get anyway. At least the GOP may get the message: "Don't tread on me!"
-- Cecil Elmore, Goose Creek, SC
There are many more uses of the slogan on the site, but there doesn't seem to be any clear consensus of what they've decided to claim it really means.