Objective To investigate the effect of eplerenone on the expression of matrix metalloproteinase-2 (MMP-2) in cardiac fibroblasts stimulated by high glucose and interleukin (IL)-1β. Methods Human cardiac fibroblasts were cultured in fluid with normal glucose (normal control group), high glucose (high glucose group) and iso-osmotic mannitol (hyperosmotic control group) in the absence or presence of IL-1β and/or eplerenone. The activity of MMP-2 in the supernatant was assessed by gel zymography, and the expression of MMP-2 mRNA and tissue inhibitor of metalloproteinase-2 (TIMP-2) mRNA was determined by RT-PCR. Results The activity of MMP-2 and MMP-2 mRNA in high glucose group and hyperosmotic control group were significantly higher than those in normal control group (P<0.01, P<0.05), while the activity of MMP-2 and MMP-2 mRNA in hyperosmotic control group were significantly lower than those in high glucose group (P<0.05). There was a twofold increase in the activity of MMP-2 and expression of MMP-2 mRNA in high glucose+IL-1β group as compared with high glucose group. The activity of MMP-2 and expression of MMP-2 mRNA in high glucose+eplerenone group and normal control+IL-1β+eplerenone group were significantly lower than those in high glucose group and normal control+IL-1β group (P<0.05). Conclusion High glucose may increase the activity of MMP-2 and expression of MMP-2 mRNA in human cardiac fibroblasts through osmotic and non-osmotic pathways, which may be enhanced by IL-1β and inhibited by eplerenone.