Thermoplastic starch plasticized with glycerol is very sensitive to humidity and aging. In this study, hyperbranched poly(1,4-butanediol citrate) is prepared via a one-step method, and is mixed with glycerol as the co-plasticizer of starch to prepare thermoplastic starch films. The structure of hyperbranched poly(1,4-butanediol citrate) is studied by performing Fourier-transform infrared spectroscopy and 1H nuclear magnetic resonance analyses. The interaction between starch and co-plasticizer, and crystallinity, mechanical properties, anti-aging properties, thermal stability, transmittance, and moisture absorption of thermoplastic starch films are studied. The results illustrate that poly(1,4-butanediol citrate)/glycerol has a synergistic effect on the plasticization, anti-aging properties, transmittance, and moisture adsorption properties of thermoplastic starch films. The thermoplastic starch film with a poly(1,4-butanediol citrate)/glycerol weight ratio of 2:28 has the maximum elongation at break, the highest transmittance, the optimal ability to inhibit the long-term retrogradation of starch and the lowest moisture content at the relative humidity of 68%. Elongation at break of thermoplastic starch film with poly(1, 4-butanediol citrate)/glycerol at 2/28 and stored for 3 and 30 days are (101.1 ± 14.0)% and (91.7 ± 2.7)%, respectively, which are 1.8 and 4.4 times that of the thermoplastic starch film with only glycerol, respectively. These phenomenon not only depend on the interaction between starch and co-plasticizer, but also may be related to the compatibility between starch and co-plasticizer. Thus, the poly(1,4-butanediol citrate)/glycerol combination has potential applications in the processing of thermoplastic starch. 1 Introduction