Adipose and endothelial dysfunction is associated with cardiovascular disease. Perivascular adipose tissue (PVAT) directly surrounds vessels and influences vessel function via a paracrine effect, and adenosine monophosphate (AMP)-activated protein kinase (AMPK) modulates the metabolic pathway, thus, the present study hypothesized that activation of AMPK in PVAT may regulate endothelial function in pathological settings. The present study investigated the effect of methotrexate (MTX) on adipocytokine expression in PVAT with an emphasis on the regulation of endothelial function. The effects of MTX and the mechanisms involved were investigated using a relaxation assay and western blot analysis. Reverse transcription-quantitative polymerase chain reaction and western blotting were used to detect the mRNA and protein expression levels. ELISA assay was used to quantify the level of TNF-alpha and IL-6. Palmitic acid (PA) stimulation induced inflammation and dysregulation of adipocytokine expression in PVAT. MTX treatment inhibited nuclear factor-kappa B p65 phosphorylation and downregulated expression of pro-inflammatory cytokines, including tumor necrosis factor-alpha and interleukin-6, whereas adiponectin expression increased. MTX increased AMPK phosphorylation under basal and inflammatory conditions in PVAT, whereas knockdown of AMPK via small interfering RNA diminished its modulatory effect, indicating that MTX inhibits inflammation in an AMPK-dependent manner. The present study prepared conditioned medium from PA-stimulated PVAT to induce endothelial dysfunction and observed that pre-treatment of PVAT with MTX effectively restored the loss of acetylcholine-induced vasodilation and increased endothelial nitric oxide synthase phosphorylation in the rat aorta. The results of the present study demonstrated that MTX ameliorated inflammation-associated adipocytokine dysregulation and thus prevented endothelial dysfunction. These data provide further pharmacological evidence regarding the beneficial effects of MTX in cardiovascular diseases.