Nitrogenase-mediated H ₂ production using heterocyst-forming cyanobacteria represents a promising approach as the process is naturally protected from O ₂-rich environments by being restricted to heterocysts. This work investigates the impact of the deletion of the vegetative cell-specific flavodiiron protein, Flv3A, on the long-term H ₂ photoproduction of the model heterocyst-forming cyanobacterium Anabaena sp. PCC 7120. The H ₂ photoproduction was evaluated in response to the presence and/or absence of N ₂ and O ₂ in the atmosphere, and in comparison to the uptake hydrogenase (Hup) deletion mutant. We demonstrate that the sole deletion of Flv3A facilitates a prolonged H ₂ photoproduction catalyzed by nitrogenase even under oxic conditions. Deletion of Flv3A did not affect the nitrogenase activity, neither its efficiency to allocate electrons to H ₂ production, nor the accumulation of sugars in the cells. Besides the transcriptional repression of hupL, the deletion of Flv3A promotes a higher O ₂ tolerance of the nitrogenase-mediated H ₂ photoproduction. Our results suggest that, in the absence of Hup, Flv3A might play a role in sustaining long-term H ₂ photoproduction under nitrogen-depleted oxic conditions. The present findings expand our understanding of nitrogenase-driven H ₂ production and offer a new possibility to overcome the current bottlenecks attained to photobiological H ₂ production in a long-term process.