Interleukin (IL)-35 is the latest member of IL-12 family, which plays an important role in other autoimmune diseases. Bone marrow mononuclear cells Coombs test‑positive hemocytopenia, also termed immunorelated hemocytopenia (IRH) is a type of autoimmune-associated diseases. The present study investigated the relationship of IL‑35 in patients with IRH. A total of 43 patients with IRH and 19 normal controls were enrolled in the current study. Serum levels of IL‑35 and IL‑17 in peripheral blood were evaluated by ELISA. Regulatory T cells (Tregs) level was detected by flow cytometry and IL‑35 subunits mRNA in Treg was determined using reverse transcription‑quantitative polymerase chain reaction: Epstein‑Barr virus induced 3 (EBI3) and IL‑12α chain p35. Effect of IL‑35 on T helper 17 cells (Th17) cells was determined by mix‑culture of IL‑35 with CD4+ T lymphocytes. Serum level of IL‑35 was decreased in untreated patients with IRH compared with remission patients (P<0.01) and was significantly associated with clinical indexes. Frequency of IL‑35 produced Tregs was lower and IL‑35 subunits mRNA in CD4+CD25+ Tregs were decreased in patients with IRH compared with health controls (P<0.01). Serum level of IL‑17 was increased in patients with IRH (P<0.01) and there was a negative correlation between IL‑35 and IL‑17 (r=‑0.553; P<0.01). The production of Th17 cells and IL‑17A mRNA expression were reduced (P<0.05) after mix‑culture of CD4+ T lymphocytes with IL‑35 compared with mix‑culture of CD4+ T lymphocytes without IL‑35. In conclusion, the present study revealed that IL‑35 may be a monitoring indicator of IRH occurrence and progression. IL‑35 level was lower and the inhibition on Th17 cells was reduced in the patients with IRH.