Harmful algal blooms (HABs) formed by toxic microalgae have seriously threatened marine ecosystems and food safety and security in recent years. Among them, Karenia selliformis has attracted the attention of scientists and society due to its acute and rapid neurotoxicity in mice. Herein, the growth and gymnodimine A (GYM-A) production of K. selliformis were investigated in diverse culture systems with different surface-to-volume (S/V) ratios and nitrogen/phosphorus concentrations. The results showed that the specific growth rates (μ), maximal cell yields, and GYM-A production levels of K. selliformis increased with higher S/V, but no significant differences were observed under different culture volumes with the same S/V, which indicated that light penetration and gas exchange in the seawater culture systems actually influenced the growth and GYM-A production of K. selliformis. The maximum cell density and photosynthetic efficiency of K. selliformis decreased under nitrogen (N) and phosphorus (P) deficiency, suggesting that the growth of K. selliformis was significantly inhibited by the deficiency in N or P. Both N and P limitation conditions, especially P deficiency, promoted the cellular GYM-A quotas of K. selliformis. In this study, a scientific basis is provided for understanding the effects of culture systems and nutrient concentrations on the growth and toxin production of K. selliformis.