A range of interactions between gut microbiota and iron (Fe) metabolism is described. Oral probiotics ameliorate host’s iron status. However, this has been proven for single-strain probiotic supplements. Dose-dependence of beneficial probiotic supplementation effect on iron turnover remains unexplored. Our study aimed to investigate the effects of oral multispecies probiotic supplementation in two doses on iron status in rats. Thirty rats were randomized into three groups receiving multispecies probiotic supplement at a daily dose of 2.5 × 109 CFU (PA group, n = 10) and 1 × 1010 CFU (PB group, n = 10) or placebo (KK group, n = 10). After 6 weeks, rats were sacrificed for analysis, blood samples, and organs (the liver, heart, kidneys, spleen, pancreas, femur, testicles, duodenum, and hair) were collected. The total fecal bacteria content was higher in the PB group vs. PA group. Unsaturated iron-binding capacity was higher in the PB group vs. KK group. Serum Fe was lower in both PA and PB vs. KK group. Iron content in the liver was higher in the PB group vs. KK group; in the pancreas, this was higher in the PB group vs. the KK and PA group, and in the duodenum, it was higher in both supplemented groups vs. the KK group. A range of alterations in zinc and copper status and correlations between analyzed parameters were found. Oral multispecies probiotic supplementation exerts dose-independent and beneficial effect on iron bioavailability and duodenal iron absorption in the rat model, induces a dose-independent iron shift from serum and intensifies dose-dependent pancreatic and liver iron uptake.