Rice possesses well-developed mechanisms of managing excessive sodium concentration under salinity stress condition. However, such mechanisms are limited in sensitive rice varieties. In the present study, we elucidated the role of riboflavin (RIB, vitamin B2) in ameliorating salinity stress in a well-known salt-sensitive rice cultivar (Oryza sativa L. cv. Koshihikari). Rice seeds were soaked in 10-μM RIB solution for 24 h, air-dried, and then the germinated seeds were cultivated hydroponically for three weeks under 25-mM NaCl to stimulate salinity stress prior to harvest. RIB-treated seedlings exhibited higher biomass accumulation, higher Sodium (Na+) and Potassium (K+) concentrations in the roots, and higher K+ concentration in the leaf blades which led to lower Na+/K+ ratio in the leaf blades. Also, RIB-treated seedlings possessed higher Calcium (Ca), Magnesium (Mg), and Copper (Cu) concentrations in the leaf blades and Iron (Fe) concentration in the roots compared to the non-treated seedlings under salinity stress. The results may be attributed to alterations in the transcript levels of Na+ transporter-encoding genes (OsHKT2;1, OsNHX1, OsNHX2, OsNHX4, OsNHX5) in the roots and leaf blades and the halotolerance gene (OsHAL3) in the leaf blades. The results indicate that RIB-treated seedlings positively regulate Na+ and K+ uptake, in turn, enhancing tissue tolerance mechanisms of alleviating salinity stress in salt-sensitive rice cultivar.