Septic shock is the most severe stage of sepsis. How immune dysregulation contributes to the pathogenesis of septic shock has not been thoroughly understood. In the current research, the phenotype and function of circulating natural killer (NK) cells of septic patients were characterised. The absolute number of NK cells was comparably reduced in septic shock survivors and non-survivors, probably owing to elevated NK cell apoptosis. Activating receptors including signalling lymphocytic activation molecule 4 (SLAMF4), natural killer cell p30-related protein (NKp30), natural killer group 2, member D (NKG2D), and DNAX accessory molecule 1 (DNAM-1) were significantly downregulated on NK cell surface in septic shock patients, especially non-survivors. Furthermore, the patients' NK cells exhibited lower expression of granzyme B and perforin, weaker target cell-induced degranulation and cytokine expression, as well as incompetent cytolytic effect. These alterations were more profound in septic shock non-survivors. Importantly, serum interleukin-35 (IL-35), which is an immunosuppressive cytokine, was remarkably elevated in septic shock patients. Besides, serum interleukin-35 concentration was positively correlated with disease scores but negatively correlated with NK cell activating receptor expression. In vitro assays indicated IL-35-induced strong suppression of NK cell activity, as evidenced by concomitant downregulation of cytokines and activating receptors along with inhibition of cytolytic capacity. Therefore, we uncovered for the first time the contributing role of IL-35 in septic shock-related human NK cell dysfunction.