Obesity and diabetes are associated with diabetic cardiomyopathy (DCM). However, the pathogenesis of DCM is not fully understood. Cannabinoid receptor gene (CNR1) has been a drug target for the treatment of obesity. Here, we reported that CNR1 expression was increased in high fat diet (HFD)-induced heart of mice. Following, the wild type (CNR1) and CNR1-knockout (CNR1) mice were employed and subjected to HFD treatments for 16 weeks to further investigate the effects of CNR1 on DCM. The results indicated that CNR1 knockout mice after HFD feeding exhibited a significant decrease of body weight and lipid accumulation in serum. Oral glucose tolerance test (OGTT) and insulin tolerance test (ITT) analysis indicated that HFD-induced insulin resistance was attenuated by CNR1 deficiency. HFD-triggered cardiac dysfunction was also improved by CNR1 knockout using echocardiographic analysis. Further, CNR1 suppression increased expressions of genes promoting fatty acid oxidation, and mitochondrial biogenesis. Also, TUNEL staining showed that CNR1 inhibition markedly reduced apoptotic levels in heart tissue sections of HFD-fed mice. Importantly, HFD-induced insulin resistance was prevented by CNR1-knockout through decreasing p-IRS1 expressions, and increasing phosphorylated insulin receptor substrate 1 (p-IRS1), phospho-AMP-activated protein kinase α (AMPKα) and phospho-acetyl-CoA carboxylase α (ACCα) expressions in heart tissue samples. In addition, CNR1 knockout impeded endoplasmic reticulum (ER) stress caused by HFD via down-regulating phospho-protein kinase-like ER kinase (PERK), phospho-eukaryotic initiation factor-2α (eIF2α), activating transcription factor 4 (ATF4) and ATF6 in heart tissue samples. Of note, we found that CNR1 knockout-improved insulin resistance, ER stress and lipid accumulation was diminished by AMPKα suppression using its inhibitor, Compound C. Therefore, the results demonstrated that therapeutic CNR1 inhibition could alleviate the progression of DCM.