Thermoplastic starch (TPS) has emerged as an essential alternative to produce environmentally friendly packaging; however, retrogradation is a disadvantage that affects its shelf life. This study analyzed the co-plasticizing effect of isosorbide on the mechanical, thermal, physicochemical, and microstructural properties and the retrogradation of films obtained by blown film extrusion from thermoplasticized starch with mixtures of glycerol and isosorbide in different ratios (3:0, 2:1, 1:2, and 0:3, respectively). The results showed that the higher concentration of isosorbide significantly increased the tensile strength; however, it reduced the elongation. Retrogradation modeled using the Avrami equation showed that the presence of isosorbide reduced the retrogradation rate (k) and modified the recrystallization mechanism (n). The relative crystallinity in the plasticized TPS films was reduced to 89%, and the adsorption significantly decreased. Isosorbide was very important in reducing the retrogradation of TPS. The best performance was obtained with the 2:1 ratio of glycerol/isosorbide due to the synergistic effect between the plasticizers. The results would allow tuning the properties of TPS films by combining glycerol/isosorbide in different ratios, which enables the design of materials tailored to potential application requirements.