Recently, microorganisms and their metabolites in the Antarctic marine environment have attracted attention as useful sources for novel therapeutics, including anticancer drugs. Here, we investigated the effects of citromycin, isolated from the Antarctic marine-derived fungus, Sporothrix sp., on human ovarian cancer cells. Citromycin inhibited the migration and invasion of human ovarian cancer SKOV3 and A2780 cells, but had no cytotoxic activity against them. Additionally, it inhibited the expression of epithelial-mesenchymal transition (EMT) markers and the activation of matrix metalloproteinase (MMP)-2 and MMP9. Moreover, extracellular signal-regulated kinase (ERK)-1/2 signaling was inhibited after citromycin treatment, and the ectopic expression of ERK negated the anti-invasive activity of citromycin. Our findings suggest that citromycin inhibits the migration and invasion of human ovarian cancer cells by downregulating the expression levels of EMT markers and MMP-2/9 via inhibition of the ERK1/2 pathway.