X-rays irradiation has been demonstrated to effectively preserve the freshness of edible fungi and delay the loss of aroma during storage. In this study, shiitake mushrooms were irradiated with X-rays dose of 0.5 kGy and stored at 2 °C for 35 days. Non-irradiated mushrooms were recorded as control group. Results indicated that 0.5 kGy X-rays treatment preserved the flavor quality by exhibiting higher volatile substance content in shiitake mushrooms. X-rays treatment promoted the activities of lipoxygenase (LOX) and alcohol dehydrogenase (ADH) and oxidation of linoleic acid. In addition, the degradation of methionine and cysteine was facilitated by X-rays. Higher enzymes activities for γ-glutamyl transpeptidase (GGT) and cystine sulfoxide lyase (CS lyase) were found in 0.5 kGy X-rays irradiated mushrooms. These findings suggest that the retention of mushroom flavor by X-rays treatment is closely related to fatty acid metabolism, sulfur-containing amino acid metabolism, and lentinic acid metabolism.