Determination of ileal endogenous amino acids (IEAAs) is necessary for the calculation of standardized ileal amino acid digestibility. This experiment was conducted to compare the response of amino acids composition of IEAAs of broilers, and digestive physiology fed the nitrogen-free diet (NFD) formulated with different ratios of dextrose to corn starch (D/CS). 28d-old broiler chickens (n = 210) with similar body weight were allocated to 5 treatment groups, including a control group (CT, basal diet, normal level of protein) and four NFD groups for a 3-days trial, designated as A (D/CS = 1.00), B (D/CS = 0.60), C (D/CS = 0.33), and D (D/CS = 0.14). The results showed that NFD significantly reduced serum IGF-1, albumin and uric acid levels when compared with the control (P < 0.05). A higher ratio of D/CS (1.00 and 0.60) increased Asp, Thr, Ser, Glu, Gly, Ala, Val, Ile, Leu, His, Tyr, Arg, and Pro contents of IEAAs when compared with the ratio of 0.33 and 0.14 (P < 0.05). Moreover, ileal DM digestibility and digestive enzyme increased with an increasing ratio of dextrose to corn starch (P < 0.001). The number of ileal goblet cells and the gene expression of Mucin 2 were higher in group A (D/CS=1.00) than in group C (D/CS = 0.33) and the control (P < 0.05). It was further observed that NFD indeed reshaped the gut microbiota, characterized by lower Bacteroidetes, a significantly increased proportion of Proteobacteria, and decreased microbial diversity (P < 0.05).Our results indicate that the chicken fed NFD were accompanied by huge digestive physiological alterations, presenting with malnutrition and accumulation of Proteobacteria in the gut. Different proportions of dextrose and starch directly affect the basal IEAAs of broiler chickens. A higher proportion of dextrose (D/CS = 1 and 0.6) in NFD increase IEAAs by promoting secretion of digestive enzyme and mucin. But the excessive proportion of starch is unsuitable for the chicken to digest NFD (D/CS = 0.14). Therefore, we suggest the ratio of dextrose to corn starch in NFD at 0.33 might be more appropriate to detect IEAAs of broiler chickens.