Objective To study the effect of macrophage-specific knockout of AMP-activated protein kinase α1 (AMPKα1) on a high fat, fructose and cholesterol diet-induced non-alcoholic fatty liver disease (NAFLD) mouse model and to explore the possible mechanism. Methods Lyz2-cre mice ( macrophage-specific cre mice) and AMPKα1^{flox / flox} (AMPK^{fl/ fl} ) mice were crossed to generate macrophage-specific AMPKα1 gene knockout ( AMPKΔMφ ) mice by DNA genotyping. AMPKα1^{flox / flox} mice and APMKΔMφ mice were fed a high fat, fructose, and cholesterol diet (CHFF diet)for 12 weeks to establish the NAFLD model. Glucose tolerance was measured by an OGTT. Differences in liver pathological changes of the two kinds of mice were observed by HE staining. Accumulation of liver lipid droplets was observed by oil red O staining. Changes in serum lipomics of two kinds of mice were assessed by GC-MS. Results Genotyping by gel electrophoresis showed that the AMPKα1^{flox / flox} band was 450 bp, the corresponding wildtype band was 334 bp, the lyz2-cre band was 700 bp, and the corresponding wildtype band was 350 bp. Western blot confirmed that macrophages of AMPK^△Mφ mice did not express AMPKα. AMPKα expression was found in macrophages of AMPK^{fl/ fl} control mice. In the NAFLD model, compared with AMPK^{fl/ fl} mice, more hepatocytes in AMPK^△Mφ mice showed steatosis and an increase in the oil red O staining area. OGTT showed that, compared with AMPK^{fl/ fl} mice, blood glucose of AMPK^△Mφ mice was increased significantly at 15 min. Non-targeted lipomics showed that the serum levels of propionic and lactic acids in AMPK^△Mφ mice were significantly lower than those in AMPK^{fl/ fl} mice. Conclusions Macrophage-specific AMPKα1 knockout promotes hepatic steatosis and downregulates serum propionic acid and lactate levels in NAFLD model mice.