Major depression disorder (MDD) has generally been found in humans worldwide. More than 350 million people suffer from MDD. The World Health Organization (WHO) predicts that MDD will be the first global burden disease in 2030. Due to undesired side effects of depression treatment, patients with depression tend to avoid medical therapies. Therefore, it is important to discover an alternative therapy from natural sources. Gastrodia elata Blume (Tian-ma) is a common Chinese medicinal herb for mind tranquilizing. Recently, some studies have reported that G. elata Bl. could be applied to ameliorate various neurodegenerative disorders. The aim of this study is to investigate the active compounds, gastrodin (GAS) and 4-hydroxybenzyl alcohol (HBA) of the water extract of G. elata Bl. (WGE), on antidepressive-like activity in rats and explore monoamine metabolism and modulated cytoskeleton remodeling-related protein expression in the Slit-Robo pathway after forced swimming test (FST). Furthermore, the analysis method of GAS using HPLC was developed and validated for source of G elata Blume identification. In vivo studies, the male Sprague Dawley rats were oral gavage with 500 mg/kg bw of WGE, 100 mg/kg bw of GAS, and 100 mg/kg bw of HBA for 21 days. It was observed that the immobility time of WGE, GAS, and HBA groups were markedly decreased in FST. Thus the depressive-like behavior could be reversed. WGE, GAS, and HBA restored higher turnover rate of serotonin and dopamine compared to control group. Due to neuronal plasticity correlates with depression, Western blot analysis was applied to Slit-Robo pathway, the neuronal cytoskeletal remodeling related protein. It was found that WGE, GAS, and HBA significantly down-regulated Slit-Robo pathway in the hippocampus that promote neuroplasticity. In vitro studies, Human oligodendroglioma (Hs683) was co-culture with WGE (0.5 mg/ml), GAS (50, 100, and 200 μM), and HBA (50, 100, and 200 μM) for 24 hours. The result indicated that WGE, GAS (50, 100, and 200 μM), and HBA (100 and 200 μM) notable down-regulated the protein expression of Slit1 directly. The validation method using HPLC was applied to investigate the quantities of GAS from preparation processes, sources and phenotypes. The results showed that the slices of Gastrodia elata f. glauca S. Chow under water extraction contained the quantity of GAS higher than G. elata Bl. f. elata. In conclusion, these results suggest that GAS provides for WGE exerting antidepressive-like effects through decreasing monoamine metabolism and down-regulating Slit-Robo pathway. Furthermore, the validation method can be used as the source of G. elata Bl. screening for health food development.