Depression is a widespread and serious mental illness. Recent evidence suggests that post-translational modifications, particularly neddylation, play a significant role in the pathophysiology of mood disorders. Neddylation, similar to ubiquitination, involves the attachment of NEDD8 to target proteins, regulating their function. However, its role in depression remains unclear. This study aimed to explore the antidepressant effects of flavokawain B (FKB), a novel neddylation inhibitor, in mouse models of depression induced by lipopolysaccharide (LPS) and chronic unpredictable mild stress (CUMS). We assessed depressive- and anxiety-like behaviors using the sucrose preference test (SPT), open-field test (OFT), forced swimming test (FST), and tail suspension test (TST). Neddylation-related protein expression (NEDD8, NAE1, UBA3, UBC12) was measured in the dorsal and ventral hippocampus (DH, VH), prelimbic cortex (PrL), and nucleus accumbens (NAc). Results showed that FKB reversed the activation of neddylation anxiety- and depressive-like behaviors in both LPS- and CUMS-treated mice. The LPS and CUMS experimental paradigms only impacted the neddylation pathway in the VH and not other brain regions (DH, PrL, IL, NAc). In conclusion, these findings suggest that FKB ameliorates depressive-like behaviors by targeting the NAE1/UBA3/UBC12 pathway in the VH, providing a potential therapeutic approach for depression.