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Objective To investigate the effect and mechanism of 6-gingerol on improving ectopic lipid accumulation in skeletal muscle of middle-aged rats induced by fructose. Methods A total of 24 male SD rats (15 months old) were randomly and equally divided into 3 groups: control group (CON, with normal drinking water), model group (FRU, with 10% fructose water) and 6-gingerol group (GIN, with 10% fructose water+0.2 mg/kg 6-gingerol by gavage). Gavage was performed once a day for consecutive 49 days. Levels of plasma glucose (GLU), triglyceride (TG) and skeletal muscle TG were measured by GOD-PAP method; lipid accumulation in skeletal muscle was detected by oil red O staining (ORO); content of plasma insulin was determined by ELISA; protein levels of AMP-activated protein kinase (AMPK), p-AMPK, acetyl-coa carboxylase (ACC), p-ACC, carnitine palmitoyltransferase 1A (CPT1A), peroxisome proliferator activated receptor alpha (PPARα) and peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC1α) in skeletal muscle were examined with Western blotting. L6 rat skeletal muscle cells were divided into 3 groups: control group (Con), model group (FRU, treated with 25 mmol/L fructose for 24 h) and 6-gingerol group (GIN, treated with 25 mmol/L fructose+10 μmol/L 6-gingerol for 24 h). TG level and lipid accumulation in L6 cells were determined by GOD-PAP and ORO staining, respectively. The expression of AMPK, p-AMPK, ACC, p-ACC and CPT1A was examined by Western blotting. Results As compared with control group, the rat model group had higher levels of plasma GLU, TG and insulin (P < 0.05), elevated TG content in both skeletal muscle tissues and cells (P < 0.05), as well as notable lipid accumulation in skeletal muscle tissues and cells (P < 0.05). Western blotting showed that the p-AMPK/AMPK ratio, p-ACC/ACC ratio, and the protein levels of CPT1A, PPARα and PGC1α were all decreased in the model group (P < 0.05). However, 6-gingerol significantly reduced the levels of plasma GLU, TG and insulin (P < 0.05), alleviated the TG contents and lipid accumulation in skeletal muscles and cells (P < 0.05), and greatly increased the p-AMPK/AMPK ratio, p-ACC/ACC ratio and the levels of CPT1A, PPARα and PGC1α in the GIN group (P < 0.05). Conclusion 6-gingerol can improve the ectopic lipid accumulation in skeletal muscle of middle-aged rats induced by fructose, which may be related to the activation of AMPK/ACC/CPT1A pathway.