<p>To investigate the effects of hesperidin on adipogenesis and the ROS/JNK/BRD4/SHP2 signaling pathway in High-Fat Diet (HFD)-induced obese rats, and to explore the potential mechanisms by which hesperidin promotes white adipose browning in obese rats. We obtained high-throughput sequencing datasets of normal-weight individuals and obese patients from the GEO database and performed bioinformatics analysis. Functional enrichment analysis, cell trajectory analysis, and intercellular communication analysis were conducted on the single-cell transcriptome sequencing data (GSM9012222, GSM9024554, GSM9012223, GSM9024555). Thirty-six male SD rats, aged six weeks, were split into two groups at random: a sham group (<i>n</i> = 9) and an obesity modelling group (<i>n</i> = 27). A diet-induced obesity model was established by feeding the rats a high-fat diet. After 12 weeks, the successfully modeled rats were randomly divided into a Model group, a Hesperidin group, and a Hesperidin+PHPS1 group, with 9 rats in each group. Following the intervention period, body weight and Lee’s index were measured, and the weights of inguinal, epididymal, and perirenal white adipose tissue (WAT), as well as interscapular brown adipose tissue (BAT), were compared among the groups. The levels of malondialdehyde (MDA), glutathione peroxidase (GPX), and superoxide dismutase (SOD) in serum and inguinal adipose tissue were measured using assay kits. Glucose homeostasis was assessed via oral glucose tolerance test (OGTT) and insulin tolerance test (ITT), which measured blood glucose levels at predefined time points. Hematoxylin-eosin (HE) staining was performed on rat inguinal white adipose tissue (iWAT). Quantitative real-time PCR (RT-qPCR) was applied to quantify the mRNA abundance of key browning-associated markers, including UCP1, PRDM16 and PGC1α. Protein expression levels were quantified via Western blot. Western blot was used to identify protein expression when 3T3-L1 cells were exposed to various stimuli in vitro. Mitochondrial membrane potential was evaluated with the JC-1 assay. Oil Red O staining was performed to visualize lipid droplets in cells. In addition, to investigate whether PHPS1 exerts effects on the rat liver, two additional rat groups were established: the control group and the PHPS1 group, with 6 rats in each group. The mRNA expression level of SHP2 was determined by RT-qPCR, and the serum activities of ALT and AST in rats were measured using corresponding assay kits. In vivo experiments showed that compared with the sham group, the model group showed increased body weight, masses of inguinal, epididymal, and perirenal WAT, serum MDA levels, and expression of NOX4, p-BRD4, PPARγ, and C/EBPα, while the mass of interscapular BAT, SOD and GPX levels in serum and adipose tissue, SOD and GPX levels, and p-SHP2 expression were decreased. The expression levels of UCP1, PRDM16 and PGC1α were downregulated. Fasting blood glucose and the area under the curve (AUC) were elevated. Meanwhile, the cell area of inguinal adipose tissue was significantly increased. Compared with the model group, Hesperidin significantly improved the above lipid metabolism, white adipose browning indicators andoxidative stress. However, PHPS1 inhibited the ameliorative effects of hesperidin on obese rats. Furthermore, PHPS1 can effectively inhibit SHP2 without exerting metabolic effects on the liver. In vitro experiments showed that hesperidin also suppressed the expression of NOX4, p-JNK, p-BRD4, PPARγ and SREBP1C, improved impaired mitochondrial function, and reduced intracellular lipid droplet formation. Bioinformatics analysis, together with in vivo and in vitro experiments, demonstrated that hesperidin inhibits high-fat diet-induced obesity by regulating the ROS/JNK/BRD4/SHP2 signaling pathway. Hesperidin inhibits HFD-induced obesity by regulating ROS/JNK/BRD4/SHP2 signaling-induced adipogenesis.</p>