Dysregulated hepatic gluconeogenesis is a hallmark of insulin resistance and type 2 diabetes mellitus (T2DM). Although existing drugs have been proven to improve gluconeogenesis, achieving this objective with functional food is of interest, especially using conjugated linoleic acid (CLA) found in dairy products. Both cis‐9, trans‐11 (c9,t11) and trans‐10, cis‐12 (t10,c12) isomers of CLA were tested in human (HepG2) and rat (H4IIE) hepatocytes for their potential effects on gluconeogenesis. The hepatocytes exposed for 24 h with 20 μM of c9,t11‐CLA had attenuated the gluconeogenesis in both HepG2 and H4IIE by 62.5% and 80.1%, respectively. In contrast, t10,c12‐CLA had no effect. Of note, in HepG2 cells, the exposure of c9,t11‐CLA decreased the transcription of gluconeogenic enzymes, cytosolic phosphoenolpyruvate carboxykinase (PCK1) by 87.7%, and glucose‐6‐phosphatase catalytic subunit (G6PC) by 38.0%, while t10,c12‐CLA increased the expression of G6PC, suggesting the isomer‐specific effects of CLA on hepatic glucose production. In HepG2, the peroxisome proliferator‐activated receptor (PPAR) agonist, rosiglitazone, reduced the glucose production by 72.9%. However, co‐administration of c9,t11‐CLA and rosiglitazone neither exacerbated nor attenuated the efficacy of rosiglitazone to inhibit glucose production; meanwhile, t10,c12‐CLA abrogated the efficacy of rosiglitazone. Paradoxically, PPARγ antagonist GW 9662 also led to 70.2% reduction of glucose production and near undetectable PCK1 expression by abrogating CLA actions. Together, while the precise mechanisms by which CLA isomers modulate hepatic gluconeogenesis directly or via PPAR warrant further investigation, our findings establish that c9,t11‐CLA suppresses gluconeogenesis by decreasing PEPCK on hepatocytes.