Atherosclerosis is a chronic inflammatory disease responsible for the majority of cases of myocardial infarction and ischemic stroke. The electronegative low-density lipoprotein, a modified subfraction of native LDL, is pro-inflammatory and plays an important role in atherogenesis. To investigate the effects of a nanoformulation (scFv anti-LDL(−)-MCMN-Zn) containing a scFv reactive to LDL(−) on the inhibition of atherosclerosis, its toxicity was evaluated in vitro and in vivo and further it was also administered weekly to LDL receptor knockout mice. The scFv anti-LDL(−)-MCMN-Zn nanoformulation did not induce cell death in RAW 264.7 macrophages and HUVECs. The 5 mg/kg dose of scFv anti-LDL(−)-MCMN-Zn did not cause any typical signs of toxicity and it was chosen for the evaluation of its atheroprotective effect in Ldlr−/− mice. This nanoformulation significantly decreased the atherosclerotic lesion area at the aortic sinus, compared with that in untreated mice. In addition, the Il1b mRNA expression and CD14 protein expression were downregulated in the atherosclerotic lesions at the aortic arch of Ldlr−/− mice treated with scFv anti-LDL(−)-MCMN-Zn. Thus, the scFv anti-LDL(−)-MCMN-Zn nanoformulation inhibited the progression of atherosclerotic lesions, indicating its potential use in a future therapeutic strategy for atherosclerosis.