Autotrophic microorganisms convert CO2 into biomass by deriving energy from light or inorganic electron donors. These CO2-fixing microorganisms have a large, but so far only partially realized, potential for the sustainable production of chemicals and biofuels. Productivities have been improved in autotrophic hosts through the introduction of production pathways and the modification of autotrophic systems by genetic engineering. In addition, approaches are emerging in which CO2 fixation pathways and energy-harvesting systems are transplanted into heterotrophic model microorganisms. Alternative promising concepts are hybrid production systems of autotrophs and heterotrophs, and bio-inorganic hybrids of autotrophic microorganisms with electrocatalysts or light-harvesting semiconductor materials. In this Review, we discuss recent advances and bottlenecks for engineering microbial autotrophy and explore novel strategies that will pave the way towards improved microbial autotrophic production platforms.