Biological wastewater treatment processes under a dynamic regime with respect to carbon substrate can result in microbial storage of internal polymers (e.g., polyhydroxybutyrate (PHB)) and their subsequent utilizations. These storage turnovers play important roles in nitrous oxide (N2O) accumulation during heterotrophic denitrification in biological wastewater treatment. In this work, a mathematical model is developed to evaluate the key role of PHB storage turnovers on N2O accumulation during denitrification for the first time, aiming to establish the key relationship between N2O accumulation and PHB storage production. The model is successfully calibrated and validated using N2O data from two independent experimental systems with PHB storage turnovers. The model satisfactorily describes nitrogen reductions, PHB storage/utilization, and N2O accumulation from both systems. The results reveal a linear relationship between N2O accumulation and PHB production, suggesting a substantial effect of PHB storage on N2O accumulation during denitrification. Application of the model to simulate long-term operations of a denitrifying sequencing batch reactor and a denitrifying continuous system indicates the feeding pattern and sludge retention time would alter PHB turnovers and thus affect N2O accumulation. Increasing PHB utilization could substantially raise N2O accumulation due to the relatively low N2O reduction rate when using PHB as carbon source.