Abstract: This study aims to investigate the underlying mechanism of 6-benzylaminopurine (6-BA) treatment on the glucosinolate (GLS) metabolism of fresh-cut broccoli. Firstly, the individual GLS and the hydrolysis products were identified from the broccoli heads. Twelve types of GLS were detected in the sample, including glucoraphanin (GRA), glucoiberin (GIB), progoitrin (PRO), gluconapin (GNA), glucoerucin (GER), glucoberteroin (GOB), glucotropaeolin (GTR), gluconasturtiin (GNS), glucobrassicin (GBR), 4-hydroxy glucobrassicin (4HGBS), 4-methoxy-glucobrassicin (4MGBS), and neoglucobrassicin (NGBS). Six hydrolysis products of GLS also included the benzonitrile, 4-isothiocyanato-1-butene, 4-pentenyl isothiocyanate, butyl-Isothiocyanate, 2-phenylethyl isothiocyanate, and sulforaphane. Among them, the GRA and sulforaphane were the main GLS and hydrolysis product in the broccoli heads. Secondly, the fresh-cut broccoli samples were treated with the 30 mg/L of 6-BA solution for ten minutes, followed by drying at room temperature for two hours, and then stored at (15 ± 1) °C for eight days. The samples treated with distilled water were used as the control. Thirdly, a systematic investigation was made to clarify the effect of 6-BA treatment on the contents of chlorophyll, GLS, and the hydrolysis products, as well as the myrosinase activity of fresh-cut broccoli. The relative expression of genes was also involved in the biosynthesis and degradation of GLS in the broccoli.The results showed that 6-BA treatment significantly delayed the yellowing of the fresh-cut broccoli by inhibiting the degradation of chlorophyll a and b. In addition, it was found that 6-BA treatment up-regulated the relative expression of genes involved in the biosynthesis of aliphatic GLS and indole GLS including MYB28, CYP79F1, CYP83A1, ST5b, and FMOGS-OX1, as well as MYB51, CYP79B2 and CYP83B1 (P<0.05).Notably, the relative expression of FMOGS-OX1, a gene coding for the enzyme responsible for the biosynthesis of GRA, was higher by 1.93 times in 6-BA-treated broccoli than in the control group on day 6. The expression level of transcription factor MYB51 in 6-BA treatment was increased by 1.41-1.91 times, and the expression of CYP79B2 was 41.90%-93.75% higher than that of control after 4 days of storage. Consequently, the contents of the main GLS of broccoli heads increased by the 6-BA treatment, including GRA, GIB, GBR, and 4MGBS. However, this treatment showed no outstanding influence on the relative expression of gene AOP2. As a result, there was no significant difference in PRO contents between the 6-BA-treated and control group. Therefore, the 6-BA treatment maintained a higher content of total GLS in the fresh-cut broccoli than that in the control group. Furthermore, the activity of myrosinase and the relative expression of myrosinase-related gene MYR in the broccoli were higher by 15.32%-90.22% and 25.86%-33.73% after four days of 6-BA treatment, respectively, compared with the control. Thus, the 6-BA treatment significantly increased the contents of hydrolysis products of GLS (P<0.05). The sulforaphane (the main isothiocyanate of broccoli) content of the fresh-cut broccoli in the 6-BA treatment was higher than that of the control group by 17.58%-25.39% than that in the control. However, the 6-BA treatment showed no obvious effect on the relative expression of ESP that was also involved in the hydrolysis of GLS. Therefore, the 6-BA treatment can be expected to maintain the high contents of GLS and isothiocyanate of fresh-cut broccoli, where the relative expression of the genes was regulated to involve in the biosynthesis and hydrolysis of GLS. These findings can provide the theoretical and technical support to obtain abundant functional components of broccoli.