This study aimed to investigate the effect and its potential mechanism of earthworm extract (EE) on pressure overload-induced cardiac fibrosis in rats. Using abdominal aortic constriction surgery, the in vivo rat model of cardiac fibrosis induced by pressure overload was established. Thirty-six Wistar male rats aged eight-week-old were randomly divided into the following groups: control group, control+EE low-dose group, model group, model+EE low-dose group, model+EE high-dose group, and model+captopril group, with six rats in each group. Rats in each group were injected intraperitoneally with EE, captopril, or vehicle daily for three weeks. Cardiac function of rats was evaluated by transthoracic echocardiography. Using the carotid artery catheterization, the blood pressure of rats was detected. Using Masson staining, picro sirius red staining and qRT-PCR, the changes of fibrosis in the heart tissues of rats were evaluated, respectively. By Western blot assay, the expression of relevant protein was detected. The results showed that compared to the control group, the hearts of the rats in the model group showed significant fibrosis (P<0.01). Compared to the model group, EE and captopril groups significantly increased the ejection fraction and fractional shortening (P<0.05, P<0.01), and significantly decreased the systolic blood pressure, diastolic blood pressure, and mean arterial pressure (P<0.05, P<0.01). EE significantly reduced collagen deposition and fibrosis in rat hearts (P<0.01) and downregulated the expression of the stromal interaction molecule 1 (STIM1), calcium-release activated calcium modulator 1 channel (Orai1), and transient receptor potential cation channel 6 (TRPC6) at protein level in the heart tissues of rats compared with model group (P<0.05, P<0.01). In conclusion, EE may ameliorate pressure overload-induced cardiac fibrosis through the regulation of the STIM1/Orai1 and TRPC6 signalings in rats.