The influence of mobility of the amorphous chains on crystallization of poly(3-hydroxybutyrate) (PHB) is detailed in this contribution. Thermal analysis of initially amorphous PHB reveals the occurrence of two distinct exotherms, indicating that cold crystallization proceeds via a two-stage process. The experimental data were rationalized taking into account the role of vitrification/devitrification of the rigid amorphous fraction (RAF) on crystallization kinetics of PHB. The rigid amorphous structure, which is established simultaneously with crystals growth during the first stage of cold crystallization, slows down crystallization before completion. The coupling between the crystalline and amorphous segments hinders further crystallization by the creation of an immobilized amorphous layer that surrounds the crystals. Further increase of the temperature results in mobilization of the RAF. Once the amorphous layer coupled with the crystals has gained sufficient mobility, chain rearrangements needed to accommodate the PHB segments into ordered structures can take place, and crystallization can proceed further. This study demonstrates that the physical state of the rigid amorphous fraction affects the crystallization kinetics of poly(3-hydroxybutyrate).