Background: Sinisan (SNS) consists of four kinds of herbs, which is the core of antidepressant prescription widely used in traditional Chinese medicine clinic treatment for depression induced by early life stress. However, the role and precise mechanism of SNS antidepressant have not yet been elucidated. Purpose: This study aimed to investigate the mechanism SNS on antidepressant of regulating mitochondrial function to improve hippocampal synaptic plasticity. Methods: 90 Sprague-Dawley (SD) rats male pups on Post-Natal Day (PND) 0 were randomly divided into Control group (ddH(2)0), Model group (ddH(2)0), Fluoxetine group (5.0 mg/kg fluoxetine), and SNS-L group (2.5 g/kg SNS), SNS-M group (5.0 g/kg SNS) and SNS-H group (10.0 g/kg SNS), 15 animals per group. Maternal separation (MS) from PND1 to PND21, drug intervention from PND60 to PND90, and behavior tests including sucrose preference test, open field test and forced swimming test from PND83 to PND90 were performed. Synaptic structure and mitochondrial structure were observed by TEM. The expression levels of PSD-95 and SYN were detected by immunohistochemistry and western blot test, the adenosine triphosphate (ATP) content in the hippocampus was detected by assay kits, and the expression levels of Mfn2, Drp1 and Fis1 protein were detected by western bolt test. Results: SNS can alleviate depression-like and anxiety-like behaviors in MS rats, improve the damage of synapses and mitochondria, reduce the decrease of ATP in hippocampus, and reverse the expression levels of PSD-95, SYN, Mfn2, Drp1, and Fis1 proteins. Conclusion: SNS reduced the risk of early life stress induced depression disorder via regulating mitochondrial function and synaptic plasticity. Targeting mitochondrial may be a novel prospective therapeutic avenue for antidepressant.