5-Fluorouracil (5-FU) is the primary chemotherapeutic agent for the clinical management of advanced gastric cancer (GC). However, the emergence of drug resistance remains an inescapable challenge. In drug-resistant cancer cells, prior treatments contribute to elevated oxidative stress, resulting in higher level of reactive oxygen species (ROS) compared to treatment-naïve cancer cells. Activation of lectin-like oxidized low-density lipoprotein receptor-1 (LOX-1) has been shown to promote ROS production and facilitate epithelial-mesenchymal transition in GC. In this study, we found that LOX-1 silencing significantly increased 5-FU sensitivity by reducing tumor cell viability and colony-forming ability. Enrichment analysis suggested that nicotinamide adenine dinucleotide phosphate oxidase 4 (NOX4) might act as a downstream effector of LOX-1, and overexpression of NOX4 was able to counteract the increased sensitivity to 5-FU induced by LOX-1 depletion. Additionally, bioinformatic predictions and in vitro experiments indicated that LOX-1 regulated NOX4 expression through JAK2/STAT3 signaling pathway. Altogether, this study provides novel evidence that LOX-1 mediated JAK2/STAT3/NOX4 axis plays a crucial role in modulating 5-FU sensitivity of GC and targeting LOX-1 may offer a promising therapeutic strategy to enhance the efficacy of 5-FU in advanced gastric cancer treatment.