(1) Liver injury caused by an overdose of acetaminophen (APAP) represents a major public health concern. Paeoniflorin (PF) has been reported to have anti-inflammatory and liver-protective effects, but the underlying mechanisms remain unclear. This study aimed to investigate the effect of PF on the crosstalk between pyroptosis and NETs in AILI. (2) APAP-treated mice were used to demonstrate the protective effect of PF on liver injury. HepG2 and dHL-60 cells were cultured to study the effects of PF on hepatocyte pyroptosis and neutrophil extracellular traps (NETs) in vitro. Moreover, cell co-culture experiments were performed, and mice were treated with a NETs-depleting agent and hepatocyte pyroptosis inhibitor to investigate the improvement of AILI induced by PF through regulating the crosstalk between hepatocyte pyroptosis and NETs. (3) PF significantly alleviated AILI. Additionally, PF inhibited the expression of pyroptosis-related proteins, high-mobility group box 1 (HMGB1), and NETs-associated proteins in vitro and in vivo. The co-culture experiments demonstrated that PF not only inhibited the NETs triggered by hepatocyte pyroptosis, but also suppressed the hepatocyte pyroptosis induced by NETs. In mice with depleted neutrophils, the level of hepatocyte pyroptosis notably decreased, indicating a diminished impact of PF. Similarly, NETs formation was reduced in mice receiving a pyroptosis inhibitor compared to the APAP group. Compared with DNase I alone, the reduction effect of PF combined with DNase I on serum ALT and AST levels decreased from 46.857% and 39.927% to 44.347% and 33.419%, respectively. Compared with DSF alone, PF combined with DSF reduced the ALT and AST levels from 46.857% and 39.927% to 45.347% and 36.419%, respectively. (4) PF demonstrated therapeutic effects on AILI. Its mechanism involves the regulation of the crosstalk between hepatocyte pyroptosis and NETs. This research substantiates the pharmacological promise of PF as a therapeutic intervention for acute AILI.