Depression poses a considerable threat to global health, underscoring the critical need for safe treatments. Because of its homology of medicine and food, Gardeniae Fructus is widely used in clinical and every-day life contexts. Gardeniae Fructus oil (OGF) is a waste byproduct of the food industry with promising recycling potential. While previous research has demonstrated the antidepressant effects of OGF, the precise mechanism remains unclear. To fill this gap in the literature, this paper elucidates the composition and potential antidepressant mechanism of OGF. According to the results, OGF is mainly composed of fatty acids and unsaponifiable matter. OGF could effectively attenuate depression-like behaviors in mice with chronic unpredictable mild stress by modulating the microbiota-gut-brain axis. Mechanisms include improving gut dysbiosis, regulating short-chain fatty acids, protecting the intestinal barrier function, reducing hippocampal damage, and alleviating neuroinflammation by inhibiting the activation of the TLR4/NF-κB/NLRP3 pathway. Fecal microbiota transplantation experiments confirmed the involvement of the gut microbiota in the modulation of depression-like behavior by OGF. This study establishes the antidepressant effects of OGF as well as the underlying mechanism, demonstrating its potential as a novel dietary intervention for depression treatment.