<p>F-53B, a substitute for perfluorooctane sulfonate (PFOS), has attracted considerable concerns due to its frequent detection in environment matrices. However, the potential health risks to mammals, especially neurodevelopmental toxicity, remain unclear. In this study, 3-week-old pubertal male rats were exposed to F-53B at concentrations of 0, 0.15, 1.5, and 15&#xa0;μg/kg for 3&#xa0;weeks continuously. Diminished cognitive abilities were observed by morris water maze (MWM) test, F-53B exposure increased the escape latency and decreased the time spent in the target quadrant of rats. Furthermore, F-53B significantly altered neurotransmitter levels in the hippocampus. Molecular docking studies indicated that F-53B might bind to metabotropic glutamate receptor 5 (mGluR5), potentially entering neurons and causing further neurotoxicity. qRT-PCR and western blot analyses were used to assess the expression of genes and proteins related to calcium pathways. Results revealed that F-53B exposure downregulated mRNA expression of ryanodine receptors (RyRs) and the phosphorylation of inositol trisphosphate receptors (IP3Rs), while upregulating sarco/endoplasmic reticulum Ca²⁺-ATPase2 (SERCA2) levels. F-53B inhibits the IP3/Ca²⁺ signaling pathway in the rat hippocampus, which may affect ER Ca²⁺ storage and release functions. Additionally, F-53B reduced the phosphorylation of IP3R, Ca²⁺/calmodulin-dependent protein kinase II (CaMKII), extracellular signal-regulated kinase 1 and 2 (ERK1/2), and cAMP response element binding protein (CREB), potentially impairing synaptic plasticity and long-term potentiation (LTP), leading to learning and memory deficits. This study reveals that F-53B induced neurodevelopmental toxicity linked to calcium pathway disruption and provides new insight into the potential long-term hazards of F-53B.</p>