Objective To explore the changes in the expression of CX3CR1 in natural killer (NK) cells from peripheral blood mononuclear cells (PBMC) in sepsis patients and its association with gut microbiota. Methods A total of 24 sepsis patients were selected from January 2020 to January 2021 at Zhongshan Hospital, Fudan University, and 10 healthy volunteers were recruited in January 2021 as healthy controls. Fecal samples and peripheral blood were collected from sepsis patients on the first and fourth days of hospitalization. Sequencing of the V3-4 region of the 16S rDNA gene of gut microbiota was performed using the Illumina MiSeq platform. The peripheral blood samples were isolated by positively selected magnetic beads, and the CD3^－CD56^＋NK cells were identified by flow cytometry. The mRNA expression of CX3CR1 was detected by qPCR, and the changes in CX3CR1 expression and its correlation with gut microbiota were analyzed. Results Compared with healthy control group, the Shannon diversity index of the gut microbiota and the proportion of Firmicutes in sepsis patients decreased; compared with admission day, the Shannon diversity of the gut microbiota in sepsis patients on the fourth day of hospitalization significantly decreased, the proportion of Proteobacteria on phylum level and the relative abundance of Enterococcus and Klebsiella on genus level significantly increased. The CX3CR1 expression of PBMC-NK cells in sepsis patients on the fourth day was significantly lower than that on the admission day (P＜0.001). Compared with surviving patients, CX3CR1 expression in non-surviving patients significantly decreased on the fourth day (P＜0.05). Spearman correlation analysis showed that CX3CR1 expression of PBMC-NK cells was positively correlated with the quantity of gut microbiota and the Shannon diversity index (P＜0.01). Conclusions The expression of CX3CR1 in PBMC-NK cells in sepsis patients decreases with disease progression, and is related to prognosis. Furthermore, its expression is found to be closely related to the gut microbiota.