Fatty acid (FA)–dependent mitochondrial activities of atrial myocardium in hypertension (HTN) and its regulation by nitric oxide (NO) remain unidentified. Here, we have studied palmitic acid (PA) regulation of cardiac mitochondrial oxygen consumption rate (OCR) in left atrial (LA) myocardium of sham and angiotensin II–induced HTN rats and their regulations by endothelial NO synthase (eNOS) and neuronal NO synthase (nNOS). The effects were compared with those of left ventricular (LV) myocytes. Our results showed that OCR was greater in HTN-LA compared with that in sham-LA. PA increased OCR in sham-LA, sham-LV, and HTN-LV but reduced it in HTN-LA. Inhibition of nNOS (S-methyl-l-thiocitrulline, SMTC) or eNOS/nNOS (Nω-nitro-l-arginine methyl ester hydrochloride, l-NAME) reduced PA increment of OCR in sham-LA but exerted no effect on OCR in HTN-LA. SMTC reduced OCR in HTN-LV and l-NAME reduced OCR in sham-LV. nNOS was the predominant source of NO in LA and LV. nNOS-derived NO was increased in HTN-LA and HTN-LV. PA reduced eNOSSer1177, nNOSSer1417, and NO level in HTN-LA but exerted no effect in sham-LA. In contrast, PA increased NO in HTN-LV and enhanced nNOSSer1417 but reduced NO level in sham-LV without affecting eNOSSer1177, eNOSThr495, or nNOSSer1417. 2-Bromopalmitate (2BP), which blocks the S-palmitoylation of target proteins, prevented PA-dependent decrease of nNOSSer1417 and OCR in HTN-LA. In HTN-LV, 2BP prevented PA-induced OCR without affecting nNOSSer1417. Our results reveal that FA-induced mitochondrial activity in atrial myocardium is impaired in HTN which is mediated by reduced nNOS activity and NO bioavailability. Metabolic dysregulation may underlie diastolic dysfunction of atrial myocardium in HTN.