Atrial fibrosis occurs frequently with structural heart disease and is considered as a major cause of arrhythmia. Microarray-based profiling predicted the differential expression of SPP1 in atrial fibrosis. Herein, we aimed to analyze the role of shRNA-mediated SPP1 knockdown in the progression of atrial fibrosis as well as the downstream mechanism. In vivo model in mice and in vitro HL-1 cell model of atrial fibrosis were developed by the angiotensin II (Ang II) method, where SPP1 expression was validated by RT-qPCR. Gain- and loss-of-function experiments were performed in Ang II-induced mice and HL-1 cells to evaluate the effect of the SPP1/TGF-β/SREBP2/PCSK9 axis on cell viability, apoptosis, collagen production and mitochondrial DNA (mtDNA) damage in atrial fibrosis. Expression of SPP1, TGF-β, SREBP2 and PCSK9 was increased in Ang II-induced mice and HL-1 cells. Silencing of SPP1 inhibited the occurrence of atrial fibrosis, as reflected by attenuated cell viability and collagen production as well as increased cell apoptosis. Conversely, upregulated SPP1 enhanced atrial fibrosis, which was related to upregulation of TGF-β. In addition, TGF-β elevated the expression of SREBP2, which promoted mtDNA damage and the consequent atrial fibrosis by augmenting the expression of PCSK9. This study uncovers previously unrecognized pro-fibrotic activities of SPP1 in atrial fibrosis, which is achieved through activation of the TGF-β/SREBP2/PCSK9 signaling pathway and promotion of mtDNA damage.