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Parkinson's disease (PD) is a progressive neurodegenerative disorder that occurs after Alzheimer's disease. Rotenone is a neurotoxin commonly used in creating PD models. Safflower (Carthamus tinctorius L.) contains some flavonoids that are effective against neurodegenerative diseases, and it has long been used in the treatment of cerebrovascular diseases in China. In this study, we investigated the preventive effect of safflower standardized flavonoid extract (SAFE) on a rotenone-induced PD rat model. The results showed that SAFE (17.5, 35, or 70 mg kg−1·day−1) treatment modified the progressive loss in body weight, alleviated behavioral deficits, and promoted survival, especially in the middle-dose SAFE (35 mg kg−1·day−1) group. SAFE treatment significantly modifies the progressive decrease in the level of DA and its metabolites, DOPAC and HVA, 5-HT and its metabolite 5-HIAA in the St, and levels of TH-positive DA-ergic neurons in the SNpc. SAFE also inhibited the decrease in TH and DA levels and increase in Ach content in the St. SAFE (35 mg kg−1·day−1) group treatment modifying the rotenone-induced downregulation of JAK2, STAT3, and ɑ7-nAChR, and also modifying the increase in ACh in the hippocampus. SAFE preventive treatment can also partially inhibit changes in the ECS parameters associated with PD. The marker components of SAFE such as Kaempferol 3-O-rutinoside or anhydrosafflor yellow B can bind with TH, JAK2, STAT3, and ɑ7-nAChR based on molecular docking analyses. Current studies have shown that SAFE is a potential candidate for the prevention of PD. [Display omitted] • SAFE modified the rotenone exposed motor dysfunction, and neurodegeneration-associated parameters in the rats. Also modified progressive decrease in the level of DA and its metabolites, DOPAC and HVA, 5-HT and its metabolite 5-HIAA. • SAFE-modified progressive decreases the levels of TH-positive DA-ergic neurons in the SNpc. Also modified the down-regulation of JAK2, STAT3, and ɑ7-nAChR in the hippocampus and increase ACh in the hippocampus. • SAFE treatment also led to partial inhibition of PD-associated changes in extracellular space diffusion parameters t 1/2 , k′, and λ. • The marker components of SAFE can bind with TH, JAK2, STAT3, ɑ7-nAChR based on molecular docking analyses. SAFE also can bind with BBB-related proteins, including MMP-9, ALOX12, ALOX15, etc. based on molecular docking analyses. [ABSTRACT FROM AUTHOR]