Objective: Interaction of antidiabetic drug 2,4-thiazolidinedione with Human serum albumin the main plasma protein responsible for a distribution of drugs in the human circulatory system. Methods: The current study was undertaken to identify the binding mechanism of 2,4-thiazolidinedione with HSA using spectroscopic, calorimetric and molecular docking methods. Results: The fluorescence and UV-vis spectral results showed that the fluorescence quenching of HSA by 2,4-thiazolidinedione was static quenching by the formation of 2,4-thiazolidinedione-HSA complex. The binding stoichiometry of 2,4-thiazolidinedione-HSA complexes was 1:1 and the binding constants (Kb) of the complexes was in the order of ~104 M-1 at 310 K, indicating that there was a moderately strong interaction between 2,4-thiazolidinedione and HSA. Circular dichroism studies reveal that the helical content of HSA slightly changed after interaction with 2,4- thiazolidinedione. Moreover, the efficiency of energy transfer, and the distance between HSA and acceptor 2,4- thiazolidinedione, were calculated. Molecular docking results revealed 2,4-thiazolidinedione can enter into the binding pocket of domain II of HSA by mainly hydrophobic and hydrogen bonds forces. Conclusion: Binding studies of drugs with HSA are potentially useful for elucidating chemico-biological interactions that can be utilized in the drug design, pharmaceutical, pharmacology, and biochemistry fields.