Objectives: Encouraging progress has been made in application of splenectomy in the treatment of relapsed hemophagocytic lymphohistiocytosis (HLH) of unknown cause. The aim was to determine the roles of lymphocyte subpopulations and inflammatory cytokines in splenectomy. Methods: We retrospectively analyzed changes in lymphocyte subpopulations and levels of inflammatory cytokines at different time-points before and after splenectomy in the patients with relapsed HLH of unknown cause, as well as the correlations between these changes and the disease prognosis. Results: During the period from June 2006 to June 2016, we enrolled 107 patients with relapsed HLH of unknown cause, of whom 29 were treated with splenectomy. Among the 29 patients, 7 cases were non-Hodgkin lymphomas based on spleen pathology, 1 case withdrew and the remaining 21 non-lymphoma cases were available for analysis. Results showed a significant increase in both percentage of CD16(+)CD56(+) NK cells (P = 0.003) and NK cell activity (P = 0.028) at 24 wk after splenectomy compared to their baseline pre-surgery levels. We also examined seven patients for the changes in cytokine levels before and after splenectomy and found that IL-21 and IL-1 alpha decreased at 4 wk after splenectomy (P < 0.05). Seven non-lymphoma patients determined as no response to treatment (NR) prior to splenectomy had significantly longer survival (P = 0.001) compared to the 24 patients with relapsed HLH of unknown cause who were also determined as NR but not treated by splenectomy. Discussion: Splenectomy can improve clinical symptoms and survival of patients with relapsed HLH of unknown cause. The mechanism is likely related to the changes in percent NK cells and cytokines (IL-21 and IL-1 alpha) after surgery.