OBJECTIVE: Calpeptin, a calpain inhibitor with potential for treating inflammatory diseases, reduces extracellular vesicle (EV) secretion. While adipose tissue is a recognized target of calpeptin, its effects on lipid metabolism remain unknown. We investigated calpeptin's impact on fatty acid (FA) profiles and metabolic pathways in human Simpson-Golabi-Behmel Syndrome adipocytes and their EVs. METHODS: Adipocytes were treated with 25 or 50 µM calpeptin, and EVs were isolated from conditioned media (CM) by ultracentrifugation. Immortalized human hepatocytes (IHHs) were pre-treated with 0 or 400 µM palmitic acid (PA) and subsequently exposed to CM from calpeptin-treated adipocytes. Total lipid FA composition was determined by gas chromatography-mass spectrometry, and gene expression with RNA-sequencing and qPCR, followed by univariate and multivariate statistics and pathway analyses. RESULTS: Calpeptin reduced EV secretion and arachidonic acid proportions in adipocytes, while also perturbing key metabolic pathways, including those of the dietarily essential polyunsaturated FAs (PUFAs). Potential biomarker candidates associated with calpeptin included C20-22 PUFAs (adipocytes) and 23:0 (EVs). Both PA and the secretome from calpeptin-treated adipocytes induced pro-inflammatory responses in IHHs. CONCLUSION: The findings suggest that calpeptin may modulate adipocyte lipid metabolism and EV secretion, with associated inflammatory responses in hepatocytes likely mediated by adipocyte‑derived secreted factors. These observations warrant further investigation into the potential adverse effects of calpeptin in the context of metabolic diseases.