This paper aimed at understanding and rationalizing the influence of both temperature and relative humidity on the mechanical behavior of thermoplastic starch (TPS). DMA experiments revealed that water molecules impact the crosslinking network by reducing the intermolecular hydrogen bond density, resulting in a less dense entanglement network. In addition, the in-situ X-ray characterization during hydration of starch revealed structural changes, which were ascribed to conformational changes in the starch chain, due to their interaction with the uptake water molecules. Finally, the study of TPS uniaxially stretched at different temperatures and humidity showed that the mechanical behavior of TPS could be rationalized by considering the ΔT parameter, which corresponds to the temperature difference between the drawing temperature and the glass transition temperature of TPS.