The present study investigated the correlation between unexplained recurrent spontaneous abortion (URSA) with CD4+CD25+ regulatory T-cell (Treg) and killer cell immunoglobulin-like receptor (KIR)-2DL1 levels. A total of 76 URSA patients were enrolled (35 without pregnancy, Group A, and 41 with early abortion, Group B). Additionally, 30 patients who received a regular abortion as planned (Group C) and 30 healthy volunteers (Group D) were selected. Peripheral venous blood and fresh decidual tissue samples were obtained from all the patients, and flow cytometry was performed to detect CD4+CD25+Treg and Foxp3 transcription factor levels. mRNA and protein KIR-2DL1 expression levels were assayed using quantitative PCR and western blot analysis, respectively. No statistically significant differences in peripheral venous blood CD4+CD25+Treg/CD4+ and Foxp3+/CD4+CD25+Treg cell proportions were found among the groups (P>0.05). However, the decidual tissues of Group C presented significantly higher levels of both cell types versus other groups (P<0.05). No statistically significant differences were found in comparisons among Groups A, B, and D (P>0.05). In peripheral venous blood, mRNA and protein KIR-2DL1 expression levels in Group C were significantly higher than those in the other three groups (P<0.05), but again, there were no statistically significant differences among Groups A, B, and D (P>0.05). In decidual tissues, KIR-2DL1 levels were significantly higher in Group C relative to Groups A, B, and D (P<0.05). Decreased CD4+CD25+Treg counts and KIR-2DL1 expression levels were closely associated with the onset of URSA. CD4+CD25+Tregs mainly exert their effects on decidual tissues, while KIR-2DL1 can act on peripheral venous blood and decidual tissues. These may present new targets for early intervention in URSA.