Abstract Imidacloprid and nitenpyram are widely used neonicotinoid pesticides worldwide and were observed to adversely affect non-target aquatic organisms. In this study, the toxic effect of imidacloprid and nitenpyram on the brain of juvenile Chinese rare minnows (Gobiocypris rarus) was investigated by determining the oxidative stress, 8-hydroxy-2-deoxyguanosine (8-OHdG) content and acetylcholinesterase (AChE) activity. The superoxide dismutase (SOD) activities did not significantly change after long-term exposure to imidacloprid and nitenpyram. A noticeable increase of catalase (CAT) activities was observed on the brain tissues under 0.1 mg/L imidacloprid and under all nitenpyram treatments (p < 0.05). The malondialdehyde (MDA) content increased markedly under 2.0 mg/L imidacloprid and 0.1 mg/L nitenpyram treatments (p < 0.05). The glutathione (GSH) content in the brain significantly increased under 0.5 and 2.0 mg/L imidacloprid (p < 0.05). A significant decrease was observed in the mRNA levels of Cu/Zn-sod under 2.0 mg/L imidacloprid and those of cat under 0.1 and 0.5 mg/L nitenpyram (p < 0.05). The mRNA levels of gpx1 clearly decreased under 2.0 mg/L imidacloprid and under 0.1 mg/L nitenpyram (p < 0.05). The treatments of 0.1 and 0.5 mg/L nitenpyram decreased cat expression levels markedly (p < 0.05). 2.0 mg/L imidacloprid raised the 8-OHdG content. The AChE activities increased markedly under 0.5 and 2.0 mg/L imidacloprid while clearly decreasing under 2.0 mg/L nitenpyram (p < 0.05). Therefore, our results indicate that imidacloprid and nitenpyram might cause adverse effects on juvenile Chinese rare minnows brain. Notably, imidacloprid had greater impacts on juvenile rare minnows compared to nitenpyram. Highlights • Chinese rare minnows were exposed to imidacloprid and nitenpyram for 60 days to investigate the neurotoxic responses. • Imidacloprid and nitenpyram impacted the Chinese rare minnow brain tissues' oxidative stress. • A 2.0 mg/L imidacloprid exposure caused an increase in the 8-OHdG content. • Both 0.5 and 2.0 mg/L imidacloprid exposure caused an increase in the AChE activity in Chinese rare minnow brain tissues. • A 2.0 mg/L nitenpyram exposure induced the inhibition of the AChE activity in the Chinese rare minnow brain tissues. [ABSTRACT FROM AUTHOR]