Trans-10, cis-12 (t10c12) conjugated linoleic acid (CLA) reduces lipid levels in adipocytes, but the mechanisms involved are still emerging. The hypotheses of this study were that t10c12 CLA treatment activated AMP-activated protein kinase (AMPK) and that the effectiveness of a low dose of t10c12 CLA would be increased when combined with an AMPK activator. We demonstrated t10c12 CLA, directly or indirectly, activated AMPK and increased the amount of phosphorylated acetyl-CoA carboxylase (ACC) in 3T3-L1 adipocytes. Compound C, a potent inhibitor of AMPK, attenuated the phosphorylation of ACC, integrated stress response (ISR), inflammatory response, reduction in key lipogenic transcription factors, and triglyceride (TG) reduction that otherwise occurred in t10c12 CLA-treated adipocytes. Treatment of adipocytes or mice with a low dose of t10c12 CLA in conjunction with the AMPK activator metformin resulted in more TG loss than treatment with the individual chemicals. Additionally, although an inflammatory response was required for robust TG reduction, the combination of t10c12 CLA with AMPK activators had a similar TG loss with a reduced inflammatory response. A microarray analysis of the transcriptional response to either t10c12 CLA, metformin, or the combination, indicated the responses were very similar, with a correlation coefficient of 0.91 or better for genes in the ISR or lipid-related pathways. Altogether, these results support our hypotheses that t10c12 CLA activates AMPK, directly or indirectly, and that metformin increases the effectiveness of t10c12 CLA in reducing TG amounts in adipocytes.