Peroxisome proliferator-activated receptor γ coactivator-1 β (PGC-1β) is known to be a transcriptional coactivator for SREBP-1, the master regulator of hepatic lipogenesis. Here, we evaluated the role of PGC-1β in the pathogenesis of fructose-induced insulin resistance by using an antisense oligonucletoide (ASO) to knockdown PGC-1β in liver and adipose tissue. PGC-1β ASO improved the metabolic phenotype induced by fructose feeding by reducing expression of SREBP-1 and downstream lipogenic genes in liver. PGC-1β ASO also reversed hepatic insulin resistance induced by fructose in both basal and insulin-stimulated states. Furthermore, PGC-1β ASO increased insulin-stimulated whole-body glucose disposal due to a threefold increase in glucose uptake in white adipose tissue. These data support an important role for PGC-1β in the pathogenesis of fructose-induced insulin resistance and suggest that PGC-1β inhibition may be a therapeutic target for treatment of NAFLD, hypertriglyceridemia, and insulin resistance associated with increased de novo lipogenesis.