Obesity-related insulin resistance is associated with an influx of pathogenic T cells into visceral adipose tissue (VAT), but the mechanisms regulating lymphocyte balance in such tissues are unknown. Here we describe an important role for the immune cytotoxic effector molecule perforin in regulating this process. Perforindeficient mice (Prf1null) show early increased body weight and adiposity, glucose intolerance, and insulin resistance when placed on high-fat diet (HFD). Regulatory effects of perforin on glucose tolerance are mechanistically linked to the control of T-cell proliferation and cytokine production in inflamed VAT. HFD-fed Prf1null mice have increased accumulation of proinflammatory IFN-g-producing CD4+ and CD8+ T cells and M1- polarized macrophages in VAT. CD8+ T cells from the VAT of Prf1null mice have increased proliferation and impaired early apoptosis, suggesting a role for perforin in the regulation of T-cell turnover during HFD feeding. Transfer of CD8+ T cells from Prf1null mice into CD8-deficient mice (CD8null) resulted in worsening of metabolic parameters compared with wild-type donors. Improved metabolic parameters in HFD natural killer (NK) cell-deficient mice (NKnull) ruled out a role for NK cells as a single source of perforin in regulating glucose homeostasis. The findings support the importance of T-cell function in insulin resistance and suggest that modulation of lymphocyte homeostasis in inflamed VAT is one possible avenue for therapeutic intervention.