Background: Peritoneal fibrosis (PF) is a serious pathophysiology of peritoneal dialysis (PD). An ongoing focus of research is the potential fibrogenic nature of methylglyoxal (MGO) in conventional PD fluid (PDF). The aim of the current study was to explore the effects of the uremic milieu on the promotion of PF by MGO using rats with adenine-induced renal failure (RF). Methods: Adenine-treated Sprague-Dawley rats were randomly assigned to receive continuous peritoneal injections of PDF with or without MGO for three weeks or were left untreated for the same duration. Rats without RF were also assigned to three groups. The peritoneal histology and expression levels of type I collagen, transforming growth factor-β1 (TGF-β1), α-smooth muscle actin (αSMA), Snail, matrix metalloproteinase-2 (MMP-2), advanced glycation end-products (AGEs) and the receptor for AGE (RAGE) were then analyzed. Results: Peritoneal treatment with 5 m<smlcap>M</smlcap> MGO accelerated the fibrous peritoneal thickening progression promoted by exposure to standard PDF in the rats with RF, but not in the rats with a normal renal function. Treatment with MGO significantly augmented the proliferation of mesenchymal-like mesothelial cells, accumulation of AGE, de novo expression of αSMA and RAGE and gene expression of type I collagen, TGF-β1, Snail and MMP-2, whereas both MGO and RF alone had, at most, marginal effects on the changes in these biological parameters. Conclusions: In the present study, the adverse effects of MGO on the peritoneum became more prominent under conditions of a uremic milieu. These findings imply that MGO and uremia act cooperatively to induce PF.