Avermectin, a widely used insecticide, has raised widespread concern over its potential toxic risk to non-target organisms. The safety of the nervous system occupies a crucial position in pesticide risk assessment, primarily because it is highly susceptible to interference by external chemicals. Within the nervous system, the blood-brain barrier (BBB) plays an essential role in maintaining system stability and function. The Wnt/β-catenin signaling pathway is known to regulate BBB integrity as well as nervous system development. Morphological observations showed that avermectin exposure caused head edema and intracranial hemorrhage in zebrafish larvae. Meanwhile, analysis of the expression levels of key proteins involved in Wnt/β-catenin signal transduction revealed that avermectin inhibited the expression of Wnt1, Wnt3a and β-catenin, and could specifically bind to Wnt1 and β-catenin proteins, indicating that this compound has the potential to disrupt the signal transduction of this pathway. Furthermore, observation of BBB structure using the Tg(flk1:eGFP; gata1:mCherry) line demonstrated that avermectin disrupted BBB integrity, manifested as blood leakage; in the Tg(flk1:eGFP) line, microinjection of DAPI dye revealed obvious substance leakage; observation of astrocyte development using the Tg(gfap:eGFP) line showed that avermectin impaired astrocyte development. In addition, avermectin could specifically bind to tight junction proteins (Claudin-1, Claudin-5 and Occludin) and inhibit the expression of the corresponding proteins and genes. In all experiments, Wnt/β-catenin signaling pathway modulators were applied, and the results consistently indicated that pathway inhibitors aggravated BBB damage, whereas agonists alleviated the damage. This study confirms that avermectin disrupts the integrity of the BBB in zebrafish larvae, and that the Wnt/β-catenin signaling pathway participates in mediating this injury process.