Thermoplastic starch (TPS) samples have been prepared in the present study using glycerol and sorbitol as co-plasticizers. Two different amounts of plasticizer, i.e. 36 and 42 wt.%, and sorbitol/glycerol ratios of 3:0, 2:1, 1:2 and 0:3 were incorporated for plasticizing granular corn starch using a laboratory co-rotating twin screw extruder. The hydrogen bond formation between plasticizers and starch chains was investigated by Fourier Transform Infrared Spectroscopy (FT-IR). X-ray diffraction (XRD) was also employed to study the crystalline structure of TPS over a period of two months to investigate the possible retrogradation process of starch. FT-IR results illustrated that the strength of starch-plasticizer hydrogen bond enhances by increasing the total amount of plasticizer and sorbitol/glycerol ratio. XRD spectra revealed A-type crystals of native starch are turned to anhydrous Va-type crystals after gelatinization. However, retrogradation and formation of hydrated Vh-type crystals may occur in the samples over storage time. Based on XRD results, glycerol plasticized starch samples are extremely prone to retrogradation as evidenced by formation of Vh-type characteristic peak. While, in the co-plasticized starch samples the mentioned peak is weakened and totally disappeared if only sorbitol is used for plasticization. In other words, the retrogradation can be effectively suppressed in the samples plasticized with higher amount of sorbitol. This is attributed to the strong hydrogen bonding between the starch chains and the sorbitol molecules.