Inhibition of fatty acid uptake by TGR5 prevents diabetic cardiomyopathy. Diabetic cardiomyopathy is characterized by myocardial lipid accumulation and cardiac dysfunction. Bile acid metabolism plays a crucial role in cardiovascular and metabolic diseases. TGR5, a major bile acid receptor, is involved in metabolic regulation and myocardial protection. However, the role of the bile acid-TGR5 pathway in maintaining cardiometabolic homeostasis remains unclear. This study shows that decreased plasma bile acid levels are observed in participants with diabetic myocardial injury. Cardiomyocyte-specific TGR5-deleted mice on a high-fat diet and streptozotocin treatment or bred on the diabetic db/db genetic background exhibit increased myocardial lipid accumulation and cardiac dysfunction. TGR5 deletion enhances cardiac fatty acid uptake, leading to lipid accumulation. Mechanistically, TGR5 deletion promotes CD36 localization on the plasma membrane through upregulation of CD36 palmitoylation mediated by DHHC4. These findings indicate that the TGR5-DHHC4 pathway regulates cardiac fatty acid uptake, highlighting the therapeutic potential of targeting TGR5 in diabetic cardiomyopathy. TGR5 inhibition reduces fatty acid uptake and lipid accumulation in the heart. TGR5 activation prevents cardiac dysfunction and cardiolipotoxicity. TGR5 inhibits fatty acid uptake and PM localization of CD36. TGR5-DHHC4 signaling regulates CD36 palmitoylation. DHHC4 KD rescues cardiac dysfunction in TGR5-deficient mice. TGR5-biased bile acids are suppressed in participants with diabetic myocardial injury. The TGR5-DHHC4 pathway is a key mediator of protection against diabetic cardiomyopathy. TGR5 inhibition reduces cardiac lipid accumulation and pathological remodelling. TGR5 plays a protective role in diabetic cardiomyopathy by reducing cardiac lipid accumulation and pathological remodelling. TGR5 is involved in regulating calcium signalling, inflammatory responses and oxidative stress, thereby mitigating hyperglycaemia-induced damage to cardiomyocytes. TGR5 deficiency significantly contributes to cardiac lipid accumulation in diabetic mice. TGR5 activation reduces fatty acid uptake and lipid accumulation. TGR5 inhibits CD36 palmitoylation and PM localization, reducing fatty acid uptake. TGR5 inhibition promotes CD36 palmitoylation and PM localization, increasing fatty acid uptake. TGR5-DHHC4 pathway mediates CD36 palmitoylation in cardiomyocytes, regulating PM localization of CD36 and lipid uptake. TGR5 is a key mediator of protection against diabetic cardiomyopathy. TGR5 inhibition reduces cardiac lipid accumulation and pathological remodelling. TGR5 activation prevents cardiac dysfunction and cardiolipotoxicity. TGR5 is a potential therapeutic target for diabetic cardiomyopathy. TGR5 inhibition reduces fatty acid uptake and lipid accumulation in cardiomyocytes. TGR5 activationInhibition of fatty acid uptake by TGR5 prevents diabetic cardiomyopathy. Diabetic cardiomyopathy is characterized by myocardial lipid accumulation and cardiac dysfunction. Bile acid metabolism plays a crucial role in cardiovascular and metabolic diseases. TGR5, a major bile acid receptor, is involved in metabolic regulation and myocardial protection. However, the role of the bile acid-TGR5 pathway in maintaining cardiometabolic homeostasis remains unclear. This study shows that decreased plasma bile acid levels are observed in participants with diabetic myocardial injury. Cardiomyocyte-specific TGR5-deleted mice on a high-fat diet and streptozotocin treatment or bred on the diabetic db/db genetic background exhibit increased myocardial lipid accumulation and cardiac dysfunction. TGR5 deletion enhances cardiac fatty acid uptake, leading to lipid accumulation. Mechanistically, TGR5 deletion promotes CD36 localization on the plasma membrane through upregulation of CD36 palmitoylation mediated by DHHC4. These findings indicate that the TGR5-DHHC4 pathway regulates cardiac fatty acid uptake, highlighting the therapeutic potential of targeting TGR5 in diabetic cardiomyopathy. TGR5 inhibition reduces fatty acid uptake and lipid accumulation in the heart. TGR5 activation prevents cardiac dysfunction and cardiolipotoxicity. TGR5 inhibits fatty acid uptake and PM localization of CD36. TGR5-DHHC4 signaling regulates CD36 palmitoylation. DHHC4 KD rescues cardiac dysfunction in TGR5-deficient mice. TGR5-biased bile acids are suppressed in participants with diabetic myocardial injury. The TGR5-DHHC4 pathway is a key mediator of protection against diabetic cardiomyopathy. TGR5 inhibition reduces cardiac lipid accumulation and pathological remodelling. TGR5 plays a protective role in diabetic cardiomyopathy by reducing cardiac lipid accumulation and pathological remodelling. TGR5 is involved in regulating calcium signalling, inflammatory responses and oxidative stress, thereby mitigating hyperglycaemia-induced damage to cardiomyocytes. TGR5 deficiency significantly contributes to cardiac lipid accumulation in diabetic mice. TGR5 activation reduces fatty acid uptake and lipid accumulation. TGR5 inhibits CD36 palmitoylation and PM localization, reducing fatty acid uptake. TGR5 inhibition promotes CD36 palmitoylation and PM localization, increasing fatty acid uptake. TGR5-DHHC4 pathway mediates CD36 palmitoylation in cardiomyocytes, regulating PM localization of CD36 and lipid uptake. TGR5 is a key mediator of protection against diabetic cardiomyopathy. TGR5 inhibition reduces cardiac lipid accumulation and pathological remodelling. TGR5 activation prevents cardiac dysfunction and cardiolipotoxicity. TGR5 is a potential therapeutic target for diabetic cardiomyopathy. TGR5 inhibition reduces fatty acid uptake and lipid accumulation in cardiomyocytes. TGR5 activation