Objective To investigate the delaying effect of omega-3 polyunsaturated fatty acids (n-3PUFAs) on D-galactose (D-gal) induced skeletal muscle aging in rats and its possible mechanism. Methods Thirty elderly male Wistar rats were randomly divided into blank group (0.9% normal saline injection), model group (rat model of skeletal muscle aging) and n-3PUFAs group (aging model+n-3PUFAs feeding), with 10 rats in each group. The growth, body mass, mass of soleus muscle and strength of limb muscle were compared among the rats in each group to explore the effect of n-3PUFAs on muscle regeneration in aging muscle atrophy rats. Western blot was used to detect the expression levels of calcium/calmodulin-dependent protein kinase 2 (CAMKK2) and adenosine monophosphate activated protein kinase (AMPK). SPSS statistics 21.0 was adopted for statistical analysis. One-way analysis of variance was employed for multiple group comparison, and LSD-t test was performed for pairwise multiple comparisons. Results Compared with blank group of rats [(466.04±33.83) g], the body mass of model group [(403.33±25.37)g] and n-3PUFAs group [(435.72±27.32)g] reduced; compared with model group,the body mass of rats in the n-3PUFAs group increased, the differences were statistically significant (P<0.05). Compared with blank group, the time began to climb of model group rats were longer after 1 month [(2.40±0.50) vs (1.07±0.15) s]and 2 months [ (7.33±0.76) vs (1.33±0.32)s]; compared with model group, the time required for n-3PUFAs group rats were shorter after 1 month [(1.76±0.32) vs (2.40±0.50)s] and 2 months [ (4.07±0.61) vs (7.33±0.76)s], the differences were statistically significant (P<0.05). Compared with blank group, the gap between skeletal muscle cells in the model group increased, the diameter of muscle fibers decreased, and the expression of p21 and p16 proteins was upregulated; the differences were statistically significant (P<0.05). Compared with model group, the gap between skeletal muscle cells in the n-3PUFAs group decreased, the diameter of muscle fibers increased, and the expression of p21 and pl6 proteins were downregulated; the differences were statistically significant(P<0.05). Compared with blank group, the relative protein expression of CaMKK2 and p-AMPK in the model group decreased; compared with model group, the expression of CaMKK2 and phosphorylated p-AMPK relative protein in n-3PUFAs group increased; the differences were statistically significant (P<0.05). The expression level of AMPK in the three groups had no significant difference (P>0.05). Conclusion n-3PUFAs can delay skeletal muscle aging induced by D-gal in rats, and its mechanism may be related to the up-regulation of key proteins in CaMKK2/AMPK signaling pathway.