Vitreoretinal lymphoma (VRL) is a rare disease of B-cell origin with poor prognosis. Regulatory cytokines promote tumor development by suppressing antitumor immunity in several cancer types, including B-cell malignancies. To identify the regulatory cytokines associated with poor prognosis in patients with B-cell VRL, we determined the regulatory cytokines profiles in the vitreous humor of patients with VRL. This retrospective study included 22 patients with VRL, 24 with non-infectious uveitis (NIU), and 20 with idiopathic epiretinal membrane (control). Vitreous concentrations of regulatory cytokines were assessed using a cytometric beads assay and association with clinical data was examined. IL-35 and soluble IL-2 receptor α levels were significantly higher in patients with VRL and NIU than those in the control group. The 5-year overall survival (OS) rates for the group with high intravitreal IL-35 was significantly poorer than those for the group with low intravitreal IL-35, who were diagnosed with VRL at the onset (P = 0.024, log-rank test). The 5-year OS rates with intravitreal IL-35 levels above and below the median were 40.0% and 83.3%, respectively. Our results suggest that high intravitreal IL-35 levels indicate poor prognosis for patients diagnosed with B-cell VRL at the onset.