As emerging contaminants growing threat to aquatic organisms, explore effective mitigation strategies is particularly important. Our previous studies have shown that selenium-rich Bacillus subtilis can not only alleviate the cause of brain damage by perfluorohexanoic acid (PFHxA) in Carassius auratus via the intestinal axis of the brain, but its metabolites can also alleviate PFHxA toxicity. This study further explores the potential mechanism through in vitro experiments. Findings demonstrate that apoptosis caused by PFHXA is effectively reduced with the use of selenium-rich Bacillus subtilis, which operates through the BDNF/PI3K/AKT/GSK-3β signalling pathway. Furthermore, utilisation of LY294002 and LICl inhibitors provided additional confirmation of the pivotal function of this pathway in neuroprotection. Our study results emphasize the significance of the PI3K/AKT/GSK-3β signalling pathway in promoting neuronal survival. Additionally, our findings establish a novel theoretical framework for using selenium-enriched Bacillus subtilis in environmental toxicology. Selenium-enriched Bacillus subtilis can be used as a novel microecological preparation. Implementing this approach could effectively counteract neurotoxic consequences of emerging contaminants, hence safeguarding and preserving aquatic ecosystems.