Approximately one-third of global carbon-fixation occurs in an overlooked algal organelle called the pyrenoid. The pyrenoid contains the CO2-fixing enzyme Rubisco, and enhances carbon-fixation by supplying Rubisco with a high concentration of CO2. Since the discovery of the pyrenoid over 130 years ago, the molecular structure and biogenesis of this ecologically fundamental organelle have remained enigmatic. To improve our understanding of the pyrenoid, we developed a high-throughput fluorescent protein-tagging and mass-spectrometry pipeline to determine the spatial organization of the pyrenoid. We have shown that the pyrenoid is a liquid-liquid phase separated organelle formed via Rubisco condensation mediated by a low complexity repeat protein. A conserved peptide motif found in pyrenoid localized proteins enables the targeting of proteins to the pyrenoid and provides a framework for pyrenoid assembly. Finally, we have developed bioinformatic tools to identify pyrenoid components based on protein physicochemical properties across diverse algae enabling in silico pyrenoid assembly. Collectively, these new insights are guiding the engineering of the pyrenoid into higher plants with a goal to enhance crop carbon fixation efficiency.