Background: Isoniazid (INH) and rifampicin (RFP) are first-line antituberculosis drugs; however, one of the most common adverse effects of their use is hepatotoxicity. Sagittaria sagittifolia polysaccharide (SSP) is the primary component of S. sagittifolia. Our previous research has confirmed the protective effect of SSP against INH and RFP-induced hepatic injury in in vivo model system. Objectives: In this study, we aimed to investigate the protective effect and the underlying mechanisms of SSP in cultured HepG2 cells after induction with INH and RFP. Materials and Methods: The study was designed as follows: normal (without any treatment), model (injury induced by INH + RFP), and SSP group (treated with SSP followed by INH + RFP). Cell viability, catalase (CAT), superoxide dismutase (SOD), and glutathione (GSH), along with malondialdehyde (MDA), as well as alanine aminotransferase, aspartate transaminase, and lactate dehydrogenase, were assessed by corresponding kits. Real-time polymerase chain reaction and Western blot were used to evaluate the mRNA and protein expression of Bcl-2, Bax, Nrf2, and Keap1. Results: SSP had a potential protective effect as it increased the cell viability and reduced the intracellular levels of hepatic injury markers. SSP reduced the intracellular content of MDA and increased the activity of SOD, CAT, and GSH. It is noteworthy that the optimum protective effect was observed with a 0.5 mg/mL SSP at 24 h after treatment. In addition, the protection appears to be associated with its activation of nuclear factor E2-related factor 2 signaling pathway. Conclusion: SSP protected against INH + RFP-induced hepatotoxicity in HepG2 cells and this protection might via Nrf2 signaling pathway.