The mechanism of the Sappan Lignum and Angelicae Pubescentis Radix drug pair (SL-APR) in treating knee osteoarthritis (KOA) was explored through network pharmacology and molecular docking, with the predicted results subsequently verified by in vivo experiments. First, active ingredients and targets of SL-APR were screened, and a “drug-ingredient-target” network was established. Core targets and relevant signaling pathways were identified through protein-protein interaction network, GO and KEGG enrichment analysis. Then, molecular docking was employed to analyze the binding of the components to the targets. Finally, a rat KOA model induced by 6% papain was used to validate the results predicted by network pharmacology. Research showed that SL-APR alleviated joint swelling, reduced inflammation, and mitigated cartilage damage. Western blot and real-time fluorescence quantitative PCR revealed that SL-APR reduced phosphoinositide 3-kinases (PI3K) protein levels, protein kinase B (AKT) and nuclear factor kappa-B (NF-κB) phosphorylation, and inhibited PI3K, AKT, and NF-κB mRNA expression in chondrocytes. Immunohistochemistry confirmed a reduction in phosphorylated NF-κB (p-NF-κB) expression in cartilage. ELISA analysis of serum and joint homogenate showed declining levels of PI3K, phosphorylated AKT (p-AKT), tumor necrosis factor-alpha (TNF-α), and interleukin-6 (IL-6). The therapeutic effect of SL-APR on KOA was demonstrated, with results suggesting that this effect was mediated through inhibition of the PI3K/AKT/NF-κB signaling axis, in alignment with network pharmacology predictions. The study preliminarily confirmed and elucidated part of the molecular mechanisms underlying the therapeutic effect of SL-APR on KOA, while providing modern scientific insight into the traditional Chinese medicine concept of “Tongluozhitong”.