The physicochemical characteristics and antioxidant activities of Lentinula edodes cultivated with liquid spawn were compared according to the harvest cycle (1–3 cycles). There was no significant difference in moisture content. The crude lipid and crude protein contents tended to decrease according to harvest cycle. The content of β-glucan was the highest in mushrooms after one cycle. The β-glucan content in cycles 2 and 3 was 68.5% and 62.3% that of the content after cycle 1, respectively. Mineral content in the pileus and stipe was similar. The mineral content was highest in the pileus after two cycles. Composition and contents of free amino acids tended to decrease according to the harvest cycle. Contents of essential amino acids were higher in mushroom after one cycle. Total phenol content was highest in the pileus (31.96 mg/ 100 g) and the stipe (21.10 mg/100 g) after one cycle and tended to decrease with subsequent cycles. The flavonoid content was 11.13 mg/100 g after one cycle and significantly decreased according to the harvest cycle. Antioxidant activities were also highest after one cycle and significantly decreased according to the harvest cycle. When compared to L. edodes cultured for one cycle, L. edodes cultivated with liquid spawn for three cycles was considered to have similar marketability in terms of quality characteristics.