The experiment was conducted to study the feasibility of the application of silkworm pupal oil to GIFT Oreochromis niloticus feed. Seven isonitrogenous and isolipidic diets were formulated by adding 5.0% silkworm pupal oil (SPO), marine fish oil (MFO), tilapia oil (TO), pork lard (PL), chicken oil (CO), linseed oil (LO) and soybean oil (SO), respectively, to the basal diet to feed tilapia[initial body weight (47.51±0.52) g]. Each treatment contained three replicates with 20 fish per replicate and fish were fed for 56 days. The results showed that the final body weight, weight gain rate and specific growth rate of LO and SO groups were significantly higher than that of SPO and MFO groups, but showed no significant differences with that of TO, PL and CO groups. Feed conversion ratio of SPO group was significantly higher than that of other groups, except that of MFO group, while protein efficiency rate showed an opposite trend. Feed intake showed the lowest value in SPO group, and highest value in SO group. TO group presented the lowest viscerosomatic index, body length/body depth and intraperitoneal fat ratio and the highest condition factor. Hepatosomatic index in PL group was significantly higher than that of other groups except LO group. Intraperitoneal fat ratio in MFO group was significantly higher than that of other groups except CO group. Compared with the other groups, SPO could reduce the crude lipid in whole body lipid and tissue and triglyceride (TG), glucose and malonaldehyde contents in serum. MFO could reduce the crude lipid in whole body and enhance the acetyl-CoA carboxylase (ACC) activity in fish liver. PL groups presented lower TG and total cholesterol (TC) contents in serum and higher carnitine acyl transferase-I and ACC activities in liver. The highest TG, TC, low density lipoprotein cholesterol (LDL-C) contents and aspartate transaminase activity were observed in serum of CO group. LO and SO could significantly increase the lipid retention ratio and crude lipid content in whole body and muscle. SO significantly increased the high density lipoprotein cholesterol (HDL-C) and reduced LDL-C and TC contents in serum. SPO, LO and SO, respectively, could significantly increase highly unsaturated fatty acids, α-linolenic acid and linoleic acid contents in fish muscle. In conclusion, although fish growth performance is affected, SPO has advantages of decreasing body lipid, blood lipid and glucose, protecting fish health, and optimizing muscle fatty acids composition.