Polyphenoloxidase (PPO), protease activities, melanosis and exudate loss of pre-cooked Pacific white shrimp (Litopenaeus vannamei) subjected to multiple freeze-thaw cycles (1, 3 and 5 cycles) using different thawing methods were studied. PPO and protease activities increased as freeze-thaw cycles increased (p < 0.05). Thawing using running tap water resulted in the higher increases in both PPO and protease activities of samples, compared with thawing at 4°C when freeze-thawing cycles were higher than 1 and 3 cycles, respectively. Exudate loss increased as freeze-thawing cycles increased (p < 0.05), but thawing methods had no impact on the formation of exudates. During refrigerated storage at 4°C, pre-cooked Pacific white shrimp had the continuous increases in melanosis. Generally, melanosis was more pronounced when subjected to repeated freeze-thawing (p < 0.05). Samples thawed with running tap water showed the higher melanosis than those thawed at 4°C, especially with 3 and 5 freeze-thaw cycles and extended storage time (p < 0.05). Therefore, freeze-thawing had a detrimental effect on quality of pre-cooked Pacific white shrimp during refrigerated storage.