With support from this project, we conducted stable carbon isotope incubations with four subsurface rocks and two seafloor-exposed rocks collected from the North Pond major program site during IODP Expedition 336 in 2011 and the MSM20-5 cruise in 2012, respectively. In combination with similar experiments done with basalts from the Loihi Seamount and the Juan de Fuca Ridge, our experiments document the potential for carbon fixation by basalt biofilm communities, providing the first empirical assessment of potential rates for this process. When scaled to the global production of oceanic crust, our results suggest carbon fixation rates that match earlier predictions based on thermodynamic calculations. Functional gene analyses indicate that the Calvin cycle is likely the dominant biochemical mechanism for carbon fixation in basalt-hosted biofilms. These results provide empirical evidence for autotrophy in oceanic crust, suggesting that basalt-hosted autotrophy could be a significant contributor of organic matter in this vast, dark environment.