Carnosic acid (CA), a major bioactive component in rosemary extract, has many biological and pharmaceutical activities. Smad3 acetylation can regulate the transcription of type I α2 collagen (COL1A2), which is the major component of the extracellular matrix (ECM). The aim of the current study was to evaluate whether CA inhibits COL1A2 transcription via the reduction of Smad3 acetylation against liver fibrosis. The results showed that CA treatment significantly suppressed COL1A2 transcription and markedly decreased the deposition of ECM induced by dimethylamine (DMN) in rats. Importantly, the suppression of COL1A2 transcription following CA treatment depended on the reduction of Smad3 acetylation via the activation of Sirtuin 1 (SIRT1), a nicotinamide adenine dinucleotide+ (NAD+)-dependent deacetylase. SIRT1 siRNA increased the acetylation of Smad3 and blocked CA-down-regulated Smad3 deacetylation. Notably, CA-mediated AMP-activated protein kinase-α1 (AMPKα1) activation not only increased AMPKα1 phosphorylation but also increased SIRT1 expression, thus leading to a significant reduction in Smad3 acetylation. Furthermore, CA-mediated SIRT1 activation was inhibited by AMPKα1 siRNA. Collectively, CA can inhibit the transcription of COL1A2 through SIRT1-mediated Smad3 deacetylation, and the activation of SIRT1 by CA involves the AMPKα1/SIRT1 pathway in liver fibrosis.