<p dir="ltr">Metastatic breast cancer, a critical health issue for women globally, is closely linked to aberrant lipid metabolism. One feature of this dysregulated metabolism is the increase in lipid storage in the form of lipid droplets (LDs), although what drives their accumulation or their role in metastasis is not clear. In prior studies, our laboratory demonstrated that the active form of vitamin D (1,25-dihydroxyvitamin D, 1,25(OH)₂D) downregulates pyruvate carboxylase (PC), a key metabolic enzyme, thereby reducing lipid accumulation in metastatic breast cancer cells (MCF10CA1a). Additionally, we have previously demonstrated that both 1,25(OH)₂D treatment and PC inhibition reduce metastasis. Therefore, the goal of the current research is to identify the proteins associated with LDs in metastatic MCF10CA1a breast cancer cells following 1,25(OH)₂D treatment or reduction in PC expression (shPC). Proteins from LDs and whole cell lysates (WCL) were processed for untargeted proteomic analysis. We identified significant differences in LD-associated proteins between untreated, 1,25(OH)₂D-treated, and PC-depleted cells. Notably, the hypoxia-inducible lipid droplet-associated protein (HILPDA), which has been shown to inhibit lipolysis through direct interaction with adipose triglyceride lipase (ATGL), was enriched only in the LD fraction of untreated metastatic cells. Immunofluorescence analysis revealed reduced colocalization of HILPDA with ATGL following 1,25D treatment and PC depletion, suggesting a potential factor that may contribute to diminished lipid accumulation and metastatic capacity. These findings indicate that 1,25(OH)₂D and PC depletion modulate LD dynamics and metabolic pathways essential for cancer cell progression, and highlight HILPDA as a potential therapeutic target for disrupting lipid storage and cancer progression.</p><p><br></p>