The discovery of the marine “deep biosphere”—microorganisms living deep below the seafloor—is one of the most significant and exciting discoveries since the ocean drilling program began more than 40 years ago. Study of the deep biosphere has become a research frontier and a hot spot both for geological and biological sciences. Here, we introduce the history of the discovery of the deep biosphere, and then we describe the types of environments for life below the seafloor, the energy sources for the living creatures, the diversity of organisms within the deep biosphere, and the new tools and technologies used in this research field. We will highlight several recently completed Integrated Ocean Drilling Program Expeditions, which targeted the subseafloor deep biosphere within the crust and sediments. Finally, future research directions and challenges of deep biosphere investigation towards uncovering the roles of subsurface microorganisms will be briefly addressed.