Metabolic dysfunction of the extracellular matrix (ECM) is one of the primary causes of intervertebral disc degeneration (IVDD). Previous studies have demonstrated that transcription factor Brachyury (Bry) has the potential to promote the synthesis of the collagen II and aggrecan, while the specific mechanism is still kept to be unknown. In this study, we employed a Lipopolysaccharide (LPS)-induced model of NPC degeneration and a rat acupuncture IVDD model to elucidate the precise mechanism through which Bry affects collagen II and aggrecan synthesis in vitro and in vivo. First, we confirmed Bry expression decreased in degenerated human nucleus pulposus (NP) cells (NPCs). Knockdown of Bry exacerbated the decrease of collagen II and aggrecan expression in lipopolysaccharide (LPS)-induced NPCs degeneration in vitro model. Bioinformatic analysis indicated that Smad3 may participate in the regulatory pathway on ECM synthesis regulated by Bry. Chromatin immunoprecipitation followed by quantitative polymerase chain reaction (ChIP-qPCR) and luciferase reporter gene assays demonstrated that Bry enhances the transcription of Smad3 by interacting with a specific motif on the promoter region. Additionally, western blot and reverse transcription-qPCR assays demonstrated that Smad3 positively regulates the expression of aggrecan and collagen II in NPCs. The following rescue experiments revealed that Bry-mediated regulation of ECM synthesis is partially dependent on Smad3 phosphorylation. Finally, the findings from in vivo rat acupuncture-induced IVDD model were consistent with those obtained from in vitro assays. In conclusion, this study reveals that Bry positively regulates the synthesis of collagen II and aggrecan in NP through transcriptional activation of Smad3.