The timing of the early invasion of the continents, the routes to the land, and the environmental breadth of the Cambrian explosion are important topics because they are at the core of our understanding of early evolutionary breakthroughs. Illuminating some aspects of these problems are trilobite trace fossils in tidal-flat deposits from the lower Cambrian Rome Formation in the southern Appalachian Mountains of Tennessee (USA). Morphologic details and size range of the trace fossils suggest production by olenellid trilobites, which occur as body fossils in the same unit. The occurrence of this ichnofauna, together with physical structures indicative of periodic subaerial exposure (desiccation cracks) and deposition within the intertidal zone (flat-topped ripples), shows that trilobites forayed into the upper intertidal zone during the Cambrian. Our finding supports the migration of subtidal organisms into marginal-marine, intertidal settings at the dawn of the Phanerozoic, suggesting that trilobites contributed to the establishment of the intertidal ecosystem during the Cambrian. The sequence of events involved in the colonization of early Paleozoic tidal flats is consistent with the idea that most terrestrial taxa originated from marine rather than freshwater ancestors, and that direct routes to the land from marginal-marine ecosystems were involved in the colonization of continental environments early in the Phanerozoic.