Paracetamol (APAP) is one of the most widely used non-steroidal anti-inflammatory drugs, which is frequently detected in various water bodies. Studies are limited about its toxic effects and mechanisms on non-target aquatic organisms. In this study, an estuarine bottom-dwelling fish named Mugilogobius chulae, distributed in southern China, was selected as experimental species and the changes of PXR signaling pathway, a key signaling pathway of detoxification metabolic system in liver, were investigated under APAP exposure (0.5 μg·L⁻¹, 5 μg·L⁻¹, 50 μg·L⁻¹ and 500 μg·L⁻¹) for 24 h, 72 h and 168 h. Results showed that the key genes (e.g., P-gp, MRP1, CYP1A, CYP3A, GST and SULT) and the enzymatic activities of GST, EROD and ERND in PXR signaling pathway were induced to meet the requirements of detoxification metabolism. By up-regulating the expression of GCLC gene, the reductive small molecule GSH can be rapidly synthesized to counteract the attack of free radicals produced by APAP exposure. The expressions of SIRT1 and SIRT2 proteins decreased, while the expressions of most genes in PXR signaling pathway increased. It was speculated that the expression of PXR and its downstream target genes may be regulated epigenetically by SIRT1 and SIRT2. Studies showed that the exposure to environmental relevant concentrations of APAP can affect the detoxification metabolism of non-target organisms such as Mugilogobius chulae.