Prochlorococcus is found throughout the euphotic zone in the oligotrophic open ocean. Deep mixing and sinking in aggregates or while attached to particles can, however, transport cells below this sunlit zone, depriving them of light for extended periods of time and influencing their circulation via ocean currents. Viability of these cells over extended periods of darkness could shape the ecology and evolution of the Prochlorococcus collective. We have shown that when co-cultured with a heterotrophic microbe and subjected to repeated periods of extended darkness, Prochlorococcus cells develop a heritable dark-tolerant phenotype – through an apparent epigenetic mechanism – such that they survive longer periods of darkness. Here we examine this adaptation at the level of physiology and metabolism in co-cultures of dark-tolerant and parent strains of Prochlorococcus, each grown with the heterotroph Alteromonas under diel light:dark conditions. The relative abundance of Alteromonas is higher in dark-tolerant than parental co-cultures, while dark tolerant Prochlorococcus cells are also larger, contain less chlorophyll, and are less synchronized to the light:dark cycle. Meta-transcriptome analysis of the cultures further suggests that dark-tolerant co-cultures undergo a coupled shift in which Prochlorococcus uses more organic carbon and less photosynthesis, and Alteromonas uses more organic acids and fewer sugars. Collectively, the data suggest that dark adaptation involves a loosening of the coupling between Prochlorococcus metabolism and the light:dark cycle and a strengthening of the coupling between the carbon metabolism of Prochlorococcus and Alteromonas. Overall design: To investigate dark-tolerance in Prochlorococcus, we obtained a dark-tolerant strain (as first described in Coe et al 2021) by subjecting exponentially growing co-cultures of Prochlorococcus NATL2A with Alteromonas macleodii MIT1002 on a 13:11 light:dark cycle to 3 days of darkness, and then returned them to 13:11 light:dark. Once cells resumed growth and reached late exponential growth, they were transferred into fresh media and the process was repeated until cells resumed growth within 1-2 days after dark exposure, at which point they were defined as dark-tolerant. We then examined gene expression differences in both Prochlorococcus and Alteromonas using RNA-seq for parental and dark-tolerant cell lines every 4-5h over a full 13:11 light:dark cycle. Samples include triplicate biological replicates at multiple time points over a 13:11 light:dark cycle with a repeating timepoint at sunrise.