Protein is a crucial nutrient in fish diets, and must be supplied in sufficient amounts to assure healthy growth processes and cell renewal. Feeding frequency is important to the survival and growth of fish, especially at early life stages. A few studies reported the relationship between feeding frequency and the dietary protein level, and supported that protein level required for maximum growth may be related to the feeding frequency. But how dietary protein level and feeding frequency affected growth and health of fish was still not clear. Therefore, this study was to estimate the effects of dietary protein and feeding frequency on the growth and health of fish. A 6-week feeding experiment was conducted to determine the effects of dietary protein and feeding frequency on growth, body composition, serum indexes and hepatopancreas structure of the GIFT strain of juvenile Nile tilapia,Oreochromis niloticus. Three replicate buckets of fish [average weight of (16.13±0.13) g] were fed to visual satiety with either 36.27% or 26.02% crude protein (CP) dietary, at different feeding frequencies (one meal per day, two meals per day or three meals per day). Each bucket was stocked with 15 fish. At the end of the feeding trial, with increasing dietary protein levels, there was significant increase in the final body weight (FW), weight gain rate (WGR), specific growth rate(SGR), feed efficiency(FE), condition factor(CF), the content of eviscerated whole body crude ash (EWA),  viscera moisture(VM) and the level of serum high density lipoprotein cholesterol (HDLC) of the fish, while there was remarkable decrease in protein efficiency ratio(PER), feed intake(FI),  viscerasomatic index (VSI), feed cost(FC), the content of eviscerated whole body crude fat (EWF), viscera crude fat (VF) and hepatopancreas crude fat (HF)(P < 0.05). As feeding frequency increased, FW, WGR, SGR, hepatosomatic index(HSI), CF, FC, EWF, VF, HF, the level of serum total cholesterol (TCHO), HDLC, low density lipoprotein cholesterol(LDLC), total protein(TP), triglyceride (TGK) increased significantly (P < 0.05), while FE, PER, VM, EWA hepatopancreas moisture (HM) decreased significantly(P < 0.05). Significant interactions between dietary protein level and feeding frequency were found in FW, WGR, SGR, VM and HDLC (P < 0.05). The liver cells of the fish fed with 26.02% protein dietary were swelling, vacuolated and the nucleus offset, but the cells in the fish fed with 36.27% protein dietary had distinct cytoplasm, less vacuolated. Considering all the indexes, the dietary protein content of 36.27% was fit for GIFT strain of juvenile Nile tilapia, Oreochromis niloticus, and the appropriate feeding frequency was more than 2 times/d, which can be adjusted according to the tilapia market situation.