Objective To investigate the role of Toll-like receptor 4 (TLR4) in cardiac function injury of rats with heat stroke disease by regulating myocardial ferroptosis. Methods (1) 24 SD rats were randomly divided into control group and heat stroke disease (HS) group (12 in each group). During heat attack, anal temperature of rats was measured by anal thermometer, heart rate and mean arterial pressure of rats were measured by BL-420F biological function experiment system. (2) 24 SD rats were randomly divided into control group, ferroptosis inhibitor (Liproxstatin-1) group, HS group and HS+Liproxstatin-1 group (6 in each group). Thirty minutes before modeling, rats in Liproxstatin-1 group and HS+Liproxstatin-1 group were intraperitoneally injected with Liproxstatin-1 (10 mg/kg). The expression of solute carrier family 7 member 11 (SLC7A11) in myocardial tissue was detected by immunofluorescence staining, and the expression levels of SLC7A11 and glutathione peroxidase 4 (GPX4) were detected by Western blotting. (3) 48 SD rats were randomly divided into control group, TLR4 inhibitor (TAK-242) group, HS group and HS+TAK-242 group (12 in each group). Thirty minutes before modeling, rats in TAK-242 group and HS+TAK-242 group were intraperitoneally injected with TAK-242 (3 mg/kg). The expression of TLR4 in myocardial tissue was detected by immunofluorescence staining, cardiac hemodynamic indexes [left ventricular systolic pressure (LVSP), left ventricular end-diastolic pressure (LVEDP), maximum rate of increase (+dp/dt_max) and maximum rate of decrease (–dp/dt_max) of left ventricular pressure] were detected by BL-420F biological function experiment system, cardiac function indexes [cardiac output (CO), diastolic left ventricular posterior wall thickness (LVPWd) and systolic left ventricular posterior wall thickness (LVPWs)] were detected by cardiac ultrasound, the expression levels of TLR4/NF-κB-p53 signaling pathway protein [TLR4, nuclear factor kappa B (NF-κB), p53] and ferroptosis related protein (SLC7A11 and GPX4) were detected by Western blotting. Results Compared with control group, the core body temperature and heart rate of rats in HS group increased significantly after heat shock for 180 minutes (P<0.01), the mean arterial pressure decreased significantly (P<0.05). Immunofluorescence staining results showed that compared with control group, the expression of myocardial SLC7A11 in HS group significantly decreased (P<0.01); Compared with HS group, the expression of SLC7A11 in HS+TAK-242 group improved significantly (P<0.01). The results of Western blotting showed that compared with control group, the expression levels of SLC7A11 and GPX4 in myocardium of HS group decreased significantly (P<0.05); Compared with HS group, the expression levels of SLC7A11 and GPX4 in HS+Liproxstatin-1 group improved significantly (P<0.05). Immunofluorescence staining results showed that compared with control group, the expression level of myocardial TLR4 in HS group increased significantly (P<0.01); Compared with HS group, the expression level of TLR4 in HS+TAK-242 group improved significantly (P<0.01). The results of BL-420F biological function test system showed that compared with control group, LVEDP increased, LVSP decreased (P<0.01) in HS group. LVSP, LVEDP, +dp/dt_max and –dp/dt_max were improved in HS+TAK-242 group (P<0.05). Compared with control group, CO was significantly decreased, and LVPWd and LVPWs were increased in HS group (P<0.01). Compared with HS group, CO increased, and LVPWd and LVPWs decreased in HS+TAK-242 group (P<0.05). Western blotting results showed that compared with control group, the expression levels of TLR4, NF-κB and p53 in the HS group increased significantly (P<0.01), and of SLC7A11 and GPX4 decreased significantly (P<0.01); Compared with HS group, the expression levels of TLR4, NF-κB and p53 decreased significantly, and of SLC7A11 and GPX4 increased significantly (P<0.05) in HS+TAK-242 group. Conclusion Ferroptosis may exist in myocardial injury caused by heat stroke. Inhibition of TLR4 can improve cardiac function of rats with heatstroke, and the mechanism may be related to ferroptosis mediated by TLR4/NF-κB-p53/SLC7A11 signaling pathway.