Aflatoxin B1 (AFB1) is the most toxic and common among the major types of aflatoxins. AFB1 is hepatoxic and has been implicated in an increase risk of hepatocellular carcinoma, also the effect of AFs on the brain chemistry have been documented. Probiotics have health benefits and provide of powerful benefits for the body and brain. The present study was designed to reveal efficiency of Bacillus subtilis NS4182-01 against the hematological, biochemical and histopathological alterations induced by AFB1 in rats. Both of AFB1 and B. subtilis were studied on the experimental animals (rats) that were divided into 6 groups as the following: Group 1 (control) received distilled water orally. Groups 2&3 (B. subtilis treated groups) received B. subtilis orally at a dose of 0.25 and 0.50 ml, respectively. Group 4 (AFB1 treated group) orally treated with AFB1 at a dose of 2 mg/kg b.w. Groups 5&6 (AFB1 + B. subtilis group) orally treated with AFB1 and then treated with B. subtilis at two studied doses respectively. It was noticed that the hematological measurements declined and the most biochemical measurements elevated significantly (P≤0.05) in AFB1 treated group. B. subtilis restored all studied measurements towards the normal values. Moreover, the native electrophoretic protein patterns showed that the physiological alterations occurred in the native protein and lipid moiety in addition to calcium moiety of native protein patterns as a result of AFB1 treatment were represented by hiding one or more of normal protein types with existence of abnormal ones. Therefore, the similarity index (SI%) and genetic distance (GD%) values were altered with protein (SI=40.00%; GD=60.00%), lipid moiety (SI=50.00%; GD=50.00%) and calcium moiety of native protein patterns (SI=75.00%; GD=25.00%) in AFB1 treated group. Although the treatment with B. subtilis at a dose of 0.25 ml showed ameliorative effect but could not restore the physiological state of the patterns to normalcy. While B. subtilis at a dose of 0.50 ml restored integrity of these native protein patterns by restoring the absent types with hiding the abnormal ones. Therefore, this group became physiologically similar to control group (SI=100.00%; GD=0.00%). These findings were supported by results of the histopathological examination in the most target organs (liver, kidney and brain) that were affected by AFB1 and the B. subtilis restored their histopathological integrity to normal structure and maintained architecture of these organs.