Although the significance of trace fossils in paleoenvironmental reconstructions is responsible for the rapid development of ichnology, we should not forget that ichnofossils are produced by living organisms and, as such, the biological nature of trace fossils is at the core of any study on animal–substrate interactions. In this chapter, we analyze the paleobiological facet of trace fossils. In order to do so, we revise concepts from benthic ecology and paleoecology. First, we explore the concept of modes of life, addressing feeding strategy, position in relation to the substrate–water interface, and level of motility. Second, we elaborate on the different modes that organisms have to interact with and, in particular, penetrate into the substrate. Third, we look at basic locomotion and burrowing mechanisms from a historical perspective, revisiting the pioneering work of Schäfer and the synthesis by Trueman. We exemplify all these mechanisms with examples form the trace-fossil record. Finally, we close this chapter by introducing the new paradigm of movement ecology and its potential implications in ichnological studies.