Excess lipid accumulation in the heart is associated with lipotoxicity and cardiac dysfunction due to excessive fatty acid oxidation. Peroxisome proliferator-activated receptor gamma (PPAR gamma) modulates the expression of key molecules involved in the FA metabolic pathway. Cardiomyocyte-specific overexpression of PPAR gamma causes dilated cardiomyopathy associated with lipotoxicity in mice. miR-130b-3p has been shown to be downregulated in the plasma of idiopathic dilated cardiomyopathy patients, but its role in modulating cardiomyocyte lipotoxicity via PPAR gamma remains unclear. Our objective was to investigate the protective role of miR-130b-3p against palmitate-induced lipotoxicity in cardiomyocytes through the modulation of the PPAR gamma signaling pathway. Human cardiomyoblasts were treated with palmitate. Intracellular lipid accumulation and expression of PPAR gamma and its downstream targets (CD36, FABP3, CAV1, VLDLR) were analyzed. Mitochondrial oxidative stress was assessed via MitoTracker Green and Redox Sensor Red staining and expression of CPT1B and SOD2. Endoplasmic reticulum stress and apoptosis were determined by examining GRP78, ATF6, XBP1s, CHOP, and caspase-3 expression. miR-130b-3p overexpression was achieved using transfection methods, and its effect on these parameters was evaluated. Luciferase assays were used to confirm PPAR gamma as a direct target of miR-130b-3p. Palmitate treatment led to increased lipid accumulation and upregulation of PPAR gamma and its downstream targets in human cardiomyoblasts. Palmitate also increased mitochondrial oxidative stress, endoplasmic reticulum stress and apoptosis. miR-130b-3p overexpression reduced PPAR gamma expression and its downstream signaling, alleviated mitochondrial oxidative stress and decreased endoplasmic reticulum stress and apoptosis in palmitate-stimulated cardiomyoblasts. Luciferase assays confirmed PPAR gamma as a direct target of miR-130b-3p. Our findings suggest that miR-130b-3p plays a protective role against palmitate-induced lipotoxicity in cardiomyocytes by modulating the PPAR gamma signaling pathway.