Background: Previous data in humans suggest that extreme physical inactivity (EPI) affects iron metabolism differently between sexes. Our objective was to deepen the underlying mechanisms by studying rats of both sexes exposed to hindlimb unloading (HU), the reference experimental model mimicking EPI.Methods: Eight-week-old male and female Wistar rats were assigned to control (CTL) or hindlimb unloading (HU) conditions (n = 12/group). After 7 days of HU, serum, liver, spleen, and soleus muscle were removed. Iron parameters were measured in serum samples, and ICP-MS was used to quantify iron in tissues. Iron metabolism genes and proteins were analysed by RT-qPCR and Western blot.Results: Compared with control males, control females exhibited higher iron concentrations in serum (+43.3%, p < 0.001), liver (LIC; +198%, P < 0.001), spleen (SIC; +76.1%, P < 0.001), and transferrin saturation (TS) in serum (+53.3%, P < 0.001), contrasting with previous observations in humans. HU rat males, but not females, exhibited an increase of LIC (+54% P < 0.001) and SIC (+30.1%, P = 0.023), along with a rise of H-ferritin protein levels (+60.9% and +134%, respectively, in liver and spleen; P < 0.05) and a decrease of TFRC protein levels (-36%; -50%, respectively, P < 0.05). HU males also exhibited an increase of splenic HO-1 and NRF2 mRNA levels, (p < 0.001), as well as HU females (P < 0.001). Concomitantly to muscle atrophy observed in HU animals, the iron concentration increased in soleus in females (+26.7, P = 0.004) while only a trend is observed in males (+17.5%, P = 0.088). In addition, the H-ferritin and myoglobin protein levels in soleus were increased in males (+748%, P < 0.001, +22%, P = 0.011, respectively) and in females (+369%, P < 0.001, +21.9%, P = 0.007, respectively), whereas TFRC and ferroportin (FPN) protein levels were reduced in males (-68.9%, P < 0.001, -76.8%, P < 0.001, respectively) and females (-75.9%, P < 0.001, -62.9%, P < 0.001, respectively). Interestingly, in both sexes, heme exporter FLVCR1 mRNA increased in soleus, while protein levels decreased (-39.9% for males P = 0.010 and -49.1% for females P < 0.001).Conclusions: Taken together, these data support that, in rats (1) extreme physical inactivity differently impacts the distribution of iron in both sexes, (2) splenic erythrophagocytosis could play a role in this iron misdistribution. The higher iron concentrations in atrophied soleus from both sexes are associated with a decoupling between the increase in iron storage proteins (i.e., ferritin and myoglobin) and the decrease in levels of iron export proteins (i.e., FPN and FLVCR1), thus supporting an iron sequestration in skeletal muscle under extreme physical inactivity.