Symbiotic nitrogen fixation in legumes requires the exquisite regulation of the environment within the infected region of the nodule. The microaerobic environment critical for nitrogenase activity is maintained through the physical oxygen diffusion barrier of the cortex and locally the oxygen-binding protein leghemoglobin (Lb). Leghemoglobin binds and releases oxygen with heme moiety to maintain oxygen gradients inside the infected cell (IC) during nitrogen fixation. Heme binds to diverse proteins and plays critical roles in different redox reactions. However,the role and regulation of host-controlled heme production during symbiotic nitrogen fixation are not clear. Here,we identified coproporphyrinogen III oxidase plastid related 1 (CPOP1) as a key regulator of symbiotic heme biosynthesis in Lotus japonicus. CPOP1 is specifically highly expressed in nitrogen-fixing nodules,and knocking out CPOP1 alone causes leaf etiolation and dwarfism which could be recovered by the exogenous application of nitrogen source,indicating nitrogen fixation defect. The IC-specific expression of CPOP1 was directed by the −881 to −740 bp promoter region. The cpop1 mutant shows significantly increased nodule oxygen level and decreased nitrogen fixation activity compared to the wild-type. Intriguingly, bacteria proliferation is inhibited due to the down-regulation of cell division-related gene expression upon CPOP1 knockout. Our data showed that CPOP1 is essential for the microaerobic environment control of ICs and the activation of rhizobial nitrogenase required for symbiotic nitrogen fixation, through host-regulated nodule heme synthesis.