Background This study aimed to investigate the roles and mechanisms of tenofovir alafenamide fumarate (TAF)/tenofovir disoproxil fumarate (TDF) in treating liver fibrosis. Methods The effects of TAF/TDF on carbon tetrachloride (CCl4)-induced liver fibrosis in C57BL/6 wild-type or nonstructural protein 5A transactivated protein 9 (NS5ATP9) knockout mice were studied. The differentiation, activation, and proliferation of LX-2 cells after TAF/TDF treatment were tested in vitro. The expression of NS5ATP9 and activities of transforming growth factor-beta 1 (TGF beta 1)/Sekelsky mothers against decapentaplegic homolog 3 (Smad3) and NF-kappa B/NOD-like receptor pyrin domain containing 3 (NLRP3) inflammasome signaling pathways were detected in TAF/TDF-treated mice and LX-2 cells. The genes related to extracellular matrix accumulation were detected in vivo and in vitro after NS5ATP9 silencing or knockout. Results TAF/TDF significantly inhibited CCl4-induced liver fibrosis in mice, and regulated the differentiation, activation, and proliferation of hepatic stellate cells (HSCs). Furthermore, TAF/TDF suppressed the activities of TGF beta 1/Smad3 and NF-kappa B/NLRP3 inflammasome signaling pathways in vivo and in vitro. NS5ATP9 inhibited liver fibrosis through TGF beta 1/Smad3 and NF-kappa B signaling pathways. TAF/TDF upregulated the expression of NS5ATP9 in vivo and in vitro. Finally, TAF/TDF could only show marginal therapeutic effects when NS5ATP9 was silenced and knocked out in vivo and in vitro. Conclusions TAF/TDF prevented progression and promoted reversion of liver fibrosis through assembling TGF beta 1/Smad3 and NF-kappa B/NLRP3 inflammasome signaling pathways via upregulating the expression of NS5ATP9. TAF/TDF also regulated the differentiation, activation, and proliferation of HSCs. The findings provided strong evidence for the role of TAF/TDF as a new promising therapeutic strategy in liver fibrosis.