Perfluorohexanoic acid (PFHxA), a widely used emerging alternative for 8-carbon PFAAs, has been detected at a high level in the water environment. While its toxicity and environmental health risk are still largely unknown in aquatic life. The present study aimed to evaluated the possible developmental neurotoxicity induced by PFHxA exposure (0, 0.48, 2.4, and 12 mg/L for 120 h) in the zebrafish embryo. Here, both developmental endpoints, neurotransmitters concentrations, locomotor behavior were analyzed. No significant effects on mortality, malformation rate, and growth delay were detected in the low dose treatment groups except for in the high dose group (12 mg/L). A significant increase in swimming speed were noted in the 0.48 mg/L group. Other changes including neurotransmitters concentrations and green fluorescent protein (GFP) expression in Tg (HuC-GFP) zebrafish larvae were significantly increased in 12 mg/L group. Beyond that, genes related to neurodevelopment were significantly decreased in larvae. Moreover, downregulations of protein expression levels of α1-tubulin, elavl3, and gap43 were identified. These results demonstrate that the PFAAs alternative PFHxA have no significant neurodevelopmental effects on zebrafish larvae under acute low-dose exposure, while, it is important to note that PFHxA perform inhibiting effects on neurotransmitter and central nervous system under a relatively high dose. This in vivo study could provide reliable toxicity information for risk assessments of PFHxA on aquatic ecosystems. CAPSULE: PFHxA have no significant neurodevelopmental effects on zebrafish larvae under acute low-dose exposure, while exposed with relatively high-dose, could induced the alternations of neurotransmitter concentrations as well as the genes involved in the early developmental stages of zebrafish, leading to the impairment of the nervous system in zebrafish larvae.