Abstract: Objective To develop a yolk lipid-based nano co-delivery system co-loaded with podoplanin (PDPN) small interfering RNA (si-PDPN) and paclitaxel (PTX), and to explore its impact on the efficacy and mechanism of traditional chemotherapy against esophageal squamous cell carcinoma (ESCC).Methods The differential expression of PDPN between ESCC tissues and adjacent normal tissues was analyzed using the TCGA database and then validated in tissue samples. ESCC cells with silenced expression of PDPN by siRNA were used to detect changes in cell proliferation, migration, and invasion. A co-delivery drug system (EYLNs-PTX-siPDPN) was constructed by encapsulating PTX and si-PDPN into egg yolk lipid-based nanoparticles (EYLNs). The particle size and zeta potential of the system were measured using a particle size analyzer, and particle stability was monitored over 14 days. The interference efficiency of PDPN by EYLNs-PTX-siPDPN was assessed by Western blot. The effects of the co-delivery system on KYSE150 cell proliferation, apoptosis, invasion, and migration were evaluated to assess its anti-ESCC efficacy.Results PDPN expression was significantly higher in ESCC tissues and cells than in adjacent normal tissues and normal esophageal epithelial cells. Knockdown of PDPN significantly inhibited the proliferation, migration, and invasion of KYSE150 cells. The particle size and zeta potential of EYLNs-PTX-siPDPN were (84.5±1.4) nm and (-13.3±1.0) mV, respectively, and particle size remained stable at 4 ℃ for 14 days. The system effectively delivered si-PDPN into tumor cells and suppressed PDPN expression. Compared with PTX and EYLNs-PTX, EYLNs-PTX-siPDPN significantly enhanced ESCC cell apoptosis and effectively inhibited proliferation, invasion, and migration.Conclusions Conclusions The co-delivery system EYLNs-PTX-siPDPN, which simultaneously delivers paclitaxel and si-PDPN, exhibits enhanced anti-ESCC effects.