Aim To explore the effect of free fatty acid on the M1 polarization and glycolysis in mouse macrophages. The mechanism of non-alcoholic steatohepatitis (NASH) pathogenesis is clarified from the perspective of metabolism reprogramming and polarization in macrophages, which will provide a new way for prevention and treatment of NASH. Methods Oleic acid/palmitate acid (O/P) was used to treat RAW264.7 cells to establish cell model. AMPK-specific siRNA was transfected into RAW264.7 cells to knockdown AMPK protein expression. Acadesine (AICAR) was used to stimulate the activation of AMPK. The mRNA levels of M1 type molecular markers (TNF-α, IL-6 and MCP-1), M2 type molecular markers (CD206 and IL-10) and glycolysis related genes (PGM1, PGK1, GPI1, LDHA, ALDOA, GLUT1 and HK2) were detected by qPCR, and the protein levels of p-mTOR/mTOR, p-Raptor/Raptor, p-Akt/Akt, p-AMPK and AMPK were detected by Western blot. Results After treatment of RAW264.7 cells with O/P, the protein levels of p-mTOR/mTOR and p-Akt/Akt increased (P＜0.05), the protein levels of p-Raptor/Raptor, p-AMPK and AMPK decreased (P＜0.05), the mRNA levels of glucose glycolysis related genes PGM1, PGK1, GPI1, LDHA, ALDOA, GLUT1 and HK2 increased (P＜0.05), the mRNA levels of pro-inflammatory factors TNF-α, IL-6 and MCP-1 increased (P＜0.05), while the mRNA levels of anti-inflammatory factors CD206 and IL-10 decreased (P＜0.05). After inhibiting AMPK expression in RAW264.7 cells, the protein levels of p-mTOR/mTOR and p-Akt/Akt increased (P＜0.05), the protein levels of p-Raptor/Raptor, p-AMPK and AMPK decreased (P＜0.05), the mRNA levels of glucose glycolysis related genes PGM1, PGK1, GPI1, LDHA, ALDOA, GLUT1 and HK2 increased (P＜0.05), the mRNA levels of pro-inflammatory factors TNF-α, IL-6 and MCP-1 increased (P＜0.05), while the mRNA levels of anti-inflammatory factors CD206 and IL-10 decreased (P＜0.05). Activation of AMPK activity could reverse the effect of O/P on macrophage polarization and glycolysis. Conclusion Free fatty acid promotes M1 polarization and glycolysis via inhibiting AMPK pathway in mouse macrophages.