The objective of this study was to evaluate the effect of glutamate (Glu) and citrate (Cit) on the absorption and distribution of aluminum in rats. In the in vitro experiment, 18 adult male Sprague–Dawley rats (average weight of 250 ± 15 g) were randomly divided into three groups. The entire intestine was rapidly removed and cultured in prediction samples of 20 mmol AlCl3, 20 mmol AlCl3+20 mmol Cit, and 20 mmol AlCl3+20 mmol Glu, respectively. Liquid in different intestines and the intestines were obtained for Al determination. In the in vivo chronic study, 24 adult male Sprague–Dawley rats (average weight of 127 ± 10 g) were divided into four groups fed with the following diets: no Al and Glu added (control), AlCl3 (1.2 mmol), AlCl3 (1.2 mmol) + Cit (1.2 mmol), and AlCl3 (1.2 mmol) + Glu (1.2 mmol) daily for 50 days, respectively. After rat sacrifice, blood samples were obtained for biochemical analyses, and organ samples like the brain, kidney, liver, and bone were rapidly taken for Al determination. The results showed that the absorption rate of Al with the following order: duodenum > jejunum > ileum in the in vitro study and the administration of AlCl3+Cit or AlCl3+Glu resulted in significant increases in Al absorption in the three parts of the gut (duodenum, jejunum, and ileum) compared to the AlCl3 alone group based on wet weight (P < 0.05). There were no differences between the AlCl3+Cit and AlCl3+Glu groups. In the in vivo chronic study, supplementing either AlCl3 alone or AlCl3+Glu decreased food consumption significantly (P < 0.05) compared with the control group. Compared with the control group, animals fed with the AlCl3 diet monitored for red blood cell, kidney, and liver showed a higher level (P < 0.05), but did not significantly increase Al retention in the brain and bone (P > 0.05); animals fed with AlCl3+Cit diets were monitored for higher Al retention in the brain, kidney, bone, and liver (P < 0.05), while animals fed with AlCl3+Glu diets were monitored for red blood cell, brain, and kidney (P < 0.05). Compared with the AlCl3 group, simultaneous administration of AlCl3 and Glu led to a significant increase in Al retention in red blood cell, brain, and kidney (P < 0.01) while AlCl3 and Cit in the kidney and bone (P < 0.01). Simultaneous administration of AlCl3 and Cit significantly increases plasma malondialdehyde level (P < 0.05); both simultaneous administration of AlCl3 and Glu or AlCl3 and Cit led to significant decreases in superoxide dismutase level in the plasma (P < 0.05), while AlCl3 alone did not. The results indicated that both Cit and Glu enhanced Al absorption in the intestine in vitro, and Glu increased Al deposition in red blood cell, brain, and kidney in vivo.