ABSTRACT The microbiomes colonizing macroalgal surfaces orchestrate nutrient fluxes and symbiotic interactions within the algal environment. Among these communities, Planctomycetota are often dominant taxa. Although nitrogenase ( nif ) gene clusters have been identified in Planctomycetota isolates and metagenome-assembled genomes, functional validation of nitrogen fixation in pure culture has remained elusive. Moreover, the mechanisms enabling these bacteria to overcome oxygen sensitivity and fix nitrogen in algal-associated oxic niches remain unexplored. Here, we isolated Crateriforma sp. HD03, a Planctomycetota strain from the surface of Saccharina japonica (kelp), and provided the first experimental evidence of nitrogen-fixing activity in pure-cultured Planctomycetota . Strain HD03 harbors a complete nifHDKBEN gene cluster and exhibits a remarkable nitrogen fixation rate of 14.2 ± 1.5 nmol C 2 H 4 /(10⁷ cells)/h under aerobic conditions. Genomic and physiological analysis reveals a suite of adaptations that likely mitigate oxygen stress, including genes associated with biofilm formation, hopanoid lipid synthesis, FeSII protein, hydrogenase, and bacterial microcompartments. Notably, while strain HD03 demonstrates oxygen-tolerant nitrogen fixation in pure culture, co-culture experiments with kelp under a photoperiod revealed that nifH (nitrogenase reductase gene) expression peaks during the low-oxygen dark phase, indicating that HD03 utilizes diurnal rhythms to temporally separate nitrogen fixation from photosynthetic oxygen production. A genomic survey of 142 Planctomycetota strains from NCBI GenBank database and HD03 identified two distinct clades harboring complete nifHDK gene clusters, suggesting a nitrogen-fixing potential across the phylum. By bridging the gap between genomic potential and functional validation, this study establishes Planctomycetota as important but underappreciated contributors to marine nitrogen input. IMPORTANCE Planctomycetota are abundant colonizers of macroalgal surfaces, yet their role in nitrogen fixation has remained unresolved despite genomic evidence of nitrogenase (nif) genes. Until now, no functional validation of nitrogen fixation in pure-cultured Planctomycetota has been reported. Here, we isolated Crateriforma sp. HD03 from kelp and for the first time demonstrated its ability to fix nitrogen in pure culture, confirming this key metabolic potential in marine Planctomycetota . Strain HD03 overcomes oxygen stress through a combination of biofilm formation and diurnal regulation of nifH expression, allowing nitrogen fixation under aerobic conditions to cope with the algal environment’s oxic nature. Furthermore, genomic surveys revealed nitrogen fixation gene clusters across multiple Planctomycetota clades, suggesting widespread nitrogen-fixing capability in this phylum. Collectively, these findings identify Planctomycetota as important nitrogen providers in the ocean.