Aims/hypothesis: Insulin action is purportedly modulated by Drosophila tribbles homologue 3 (TRIB3), which in vitro prevents thymoma viral proto-oncogene (AKT) and peroxisome proliferator-activated receptor-? (PPAR-?) activation. However, the physiological impact of TRIB3 action in vivo remains controversial. Methods: We investigated the role of TRIB3 in rats treated with either a control or Trib3 antisense oligonucleotide (ASO). Tissue-specific insulin sensitivity was assessed in vivo using a euglycaemic-hyperinsulinaemic clamp. A separate group was treated with the PPAR-? antagonist bisphenol-A-diglycidyl ether (BADGE) to assess the role of PPAR-? in mediating the response to Trib3 ASO. Results: Trib3 ASO treatment specifically reduced Trib3 expression by 70 to 80 in liver and white adipose tissue. Fasting plasma glucose, insulin concentrations and basal rate of endogenous glucose production were unchanged. However, Trib3 ASO increased insulin-stimulated whole-body glucose uptake by 50 during the euglycaemic-hyperinsulinaemic clamp. This was attributable to improved skeletal muscle glucose uptake. Despite the reduction of Trib3 expression, AKT2 activity was not increased. Trib3 ASO increased white adipose tissue mass by 70 and expression of Ppar-? and its key target genes, raising the possibility that Trib3 ASO improves insulin sensitivity primarily in a PPAR-?- dependent manner. Co-treatment with BADGE blunted the expansion of white adipose tissue and abrogated the insulin-sensitising effects of Trib3 ASO. Finally, Trib3 ASO also increased plasma HDL-cholesterol, a change that persisted with BADGE co-treatment. Conclusions/interpretation: These data suggest that TRIB3 inhibition improves insulin sensitivity in vivo primarily in a PPAR-?-dependent manner and without any change in AKT2 activity.