Preclinical studies have proven that nanosecond pulsed electric field (nsPEF) ablation can be a safe and effective treatment for humans with unresectable liver cancer that are ineligible for thermal ablation. The concomitant activation of antitumor immunity by nsPEF can also potentially prevent tumor recurrence. However, whether nsPEF exhibits similar efficacy in a clinical setting remains to be investigated. A prospective clinical trial (clinicaltrials.govidentifier: NCT04039747) was conducted to evaluate the safety and efficacy of ultrasound (US)‐guided nsPEF ablation in 15 patients with unresectable liver cancer that were ineligible for thermal ablation. We found that nsPEF ablation was safe and produced a 12‐month recurrence‐free survival (RFS) and local RFS of 60% (9/15) and 86.7% (13/15), respectively, in the enrolled patients. Integrative proteomic and metabolomic analysis showed that sphingolipid metabolism was the most significantly enriched pathway in patient sera after nsPEF without recurrence within 8 months. A similar upregulation of sphingolipid metabolism was observed in the intratumoral mononuclear phagocytes (MNPs), rather than other immune and nonimmune cells, of an nsPEF‐treated mouse model. We then demonstrated that lymphocyte antigen 6 complex, locus C2‐positive (Ly6c2+) monocytes first differentiated intoLy6c2+monocyte‐derived macrophages with an increase in sphingolipid metabolic activity, and subsequently intoLy6c2+dendritic cells (DCs).Ly6c2+DCs communicated with CD8+T cells and increased the proportions of IFN‐γ+CD8+memory T cells after nsPEF, and this finding was subsequently confirmed by depletion of liverLy6c2+MNPs. In conclusion, nsPEF was a safe and effective treatment for liver cancer. The alteration of sphingolipid metabolism induced by nsPEF was associated with the differentiation ofLy6c2+MNPs, and subsequently induced the formation of memory CD8+T cells with potent antitumor effect.