The harmful alga Heterosigma akashiwo possesses a hybrid nitrate reductase (NR) enzyme, NR2-2/2HbN, which has the potential to convert NO to nitrate for assimilation into biomass. In previous research, NR transcription in H. akashiwo was induced by nitrate while NR activity was inhibited by ammonium. Here, the capacity of H. akashiwo to use NO in the presence of nitrate and/or ammonium was investigated to understand the regulation of NO assimilation. Continuous cultures of H. akashiwo were acclimated to growth on nitrate, ammonium, or a mixture of both. Aliquots from these cultures were spiked with 15N-labeled NO. The expression of genes involved in nitrogen assimilation was evaluated, as well as nitrate reductase activity and assimilation of 15N-labeled nitrogen into algal biomass. Results showed that NO induced expression and activity of NR, and upregulated expression of GOGAT regardless of the presence of other inorganic nitrogen sources, while GS expression decreased over time. Furthermore, 15NO uptake and assimilation was significantly higher in cultures acclimated for growth on ammonium compared to cultures acclimated for growth on nitrate alone. Assimilation of NO may provide H. akashiwo with a competitive advantage in N-poor environments or areas with elevated NO.