Our previous studies have indicated that chronic treatment with 1-[(2-dimethylamino) ethylamino]-4-(hydroxymethyl)-7-[(4-methylphenyl) sulfonyl oxy]-9H-xanthene-9-one (XNT), an angiotensin-converting enzyme 2 (ACE2) activator, reverses hypertension-induced cardiac and renal fibrosis in spontaneously hypertensive rats (SHRs). Furthermore, XNT prevented pulmonary vascular remodelling and right ventricular hypertrophy and fibrosis in a rat model of monocrotaline-induced pulmonary hypertension. The aim of this study was to determine the mechanisms underlying the protective effects of XNT against cardiac fibrosis. Hydroxyproline assay was used to measure cardiac collagen content in control and XNT-treated (200 ng kg(-1) min(-1) for 28 days) SHRs. Cardiac ACE2 activity and protein levels were determined using the fluorogenic peptide assay and Western blot analysis, respectively. Extracellular signal-regulated kinases (ERKs; p44 and p42) and angiotensin II type 1 (AT(1)) receptor levels were quantified by Western blotting. Cardiac ACE2 protein levels were ∼15% lower in SHRs compared with Wistar-Kyoto control animals (ACE2/glyceraldehyde 3-phosphate dehydrogenase ratio: Wistar-Kyoto, 1.00 ± 0.02 versus SHR, 0.87 ± 0.01). However, treatment of SHRs with XNT completely restored the decreased cardiac ACE2 levels. Also, chronic infusion of XNT significantly increased cardiac ACE2 activity in SHRs. This increase in ACE2 activity was associated with decreased cardiac collagen content. Furthermore, the antifibrotic effect of XNT correlated with increased cardiac angiotensin-(1-7) immunostaining, though no change in cardiac AT(1) protein levels was observed. The beneficial effects of XNT were also accompanied by a reduction in ERK phosphorylation (phospho-ERK/total ERK ratio: Wistar-Kyoto, 1.00 ± 0.04; control SHR, 1.46 ± 0.25; treated SHR, 0.86 ± 0.02). Our observations demonstrate that XNT activates cardiac ACE2 and inhibits fibrosis. These effects are associated with increases in angiotensin-(1-7) and inhibition of cardiac ERK signalling.