Background/Objectives: The most used antipyretic and pain relief treatment is paracetamol (acetaminophen), also known as N-acetyl-para-aminophenol (APAP). However, it is considered potentially hazardous if consumed repeatedly in large doses or over prolonged periods. This investigation explores the effectiveness of hesperidin (Hesp) and Hesp loaded on layered double hydroxide nanoparticles (Hesp-NPs) in inhibiting the progression of acute hepatotoxicity in rats induced by APAP. Methods: LDH-Hesp-NPs were prepared and characterized. Male Wistar rats were orally treated with Hesp and Hesp-NPs at the same adjusted dose (100 mg/kg) every other day for six weeks. After 2 h of the first doses of Hesp and Hesp-NPs, the rats received one oral dose of APAP (750 mg/kg). Results: Administering of Hesp and Hesp-NPs to APAP-treated rats significantly reduced oxidant parameter (malondialdehyde) and serum enzymes (ALT, AST, LDH, and ALP) associated with liver function. Antioxidant markers in the liver, such as catalase and glutathione, also increased notably. Moreover, Hesp and Hesp-NPs enhanced the mRNA expression of liver UGT1A6, IL-10, and HO-1. Conversely, the mRNA expressions of liver CYP1A1, KEAP1, TGF-β, P53, and BAX decreased. These improvements in biochemical and molecular markers were corroborated by liver histopathology. Conclusions: Hesp and Hesp-NPs protect significantly against APAP-induced hepatotoxicity in male Wistar rats. Hesp-NPs treatment was more potent. The protective effects may be mediated via modulation of APAP biotransformation, oxidative stress, inflammation and apoptosis.