An endothermic annealing peak appears in DSC heating scans of cold-crystallized poly(L-lactic acid) after annealing above the cold-crystallization temperature. The annealing peak shifts to higher temperature and its magnitude increase with increasing annealing temperature or time. Furthermore, the origin of the annealing peak and microstructural changes of poly(L-lactic acid) were investigated by temperature-modulated differential scanning calorimetry (TMDSC), Wide-angle X-ray diffraction (WAXD), small-angle X-ray scattering (SAXS) and dynamic mechanical analysis (DMA). During TMDSC scan, the observed annealing peak on the total heat flow (THF) is separated into an endothermic peak of enthalpy relaxation on the nonreversible heat flow (NHF) and a glass transition (devitrification) step on the reversible heat flow (RHF). SAXS results showed that the significant increase in long period mainly arises from the increase in the amorphous layer after annealing. The storage modulus is enhanced with increasing annealing temperature, accompany with glass transition temperature shifts to lower temperature, and the softening is also observed above annealing temperature in DMA measurements. It is showed that the annealing at elevated temperature promotes the chain mobility of the constraint amorphous phase and divides them into more orderly rigid amorphous fraction (RAF) and more disorderly mobile amorphous fraction (MAF). The above results indicate that the annealing peak is attributed to the nonreversible enthalpy relaxation of RAF, which formed by annealing process, rather than the melting of thin lamellae or imperfect crystals formed by secondary crystallization. (C) 2013 Elsevier Ltd. All rights reserved.