Objective <p>Dipeptidyl peptidase 9 (DPP9) not only regulates tumor progression and drug sensitivity, but also modifies oxidative stress mediated ferroptosis. This study aimed to investigate the effect of DPP9 inhibition on sorafenib sensitivity and its interaction with ferroptosis in hepatocellular carcinoma (HCC).</p> Methods <p>Two HCC cell lines (Huh7 and MHCC-97H) were transfected with DPP9 siRNA, followed by detection of reactive oxygen species (ROS), ferrous iron (Fe²⁺), malondialdehyde (MDA), and ferroptosis-related proteins, and treated by 0–16&#xa0;μM sorafenib to calculate half-maximal inhibitory concentration (IC₅₀) for sensitivity assessment. Moreover, ferrostatin-1 (Fer-1) was added with or without DPP9 siRNA, followed by the above detections.</p> Results <p>Inhibition of DPP9 improved sorafenib sensitivity reflected by a lower sorafenib IC₅₀ value, and it increased ROS fluorescence intensity, Fe²⁺ level, and MDA level, which also upregulated ACSL4 expression but downregulated NRF2 and SLC7A11 expressions. Fer-1 treatment decreased ROS fluorescence intensity, Fe²⁺ level, MDA level, and reduced sorafenib sensitivity reflected by a higher sorafenib IC₅₀ value. Moreover, Fer-1 treatment weakened the effect of DPP9 inhibition on ROS fluorescence intensity, Fe²⁺ level, MDA level, most of the ferroptosis-related proteins, and sorafenib sensitivity reflected by sorafenib IC₅₀ value.</p> Conclusion <p>Inhibition of DPP9 improves sorafenib sensitivity by promoting ferroptosis in HCC, which provides novel evidence for DPP9 as an HCC treatment target synergizing with sorafenib.</p>