Objective. The sphingosine kinase 1 (SphK1)/sphingosine-1-phosphate (S1P) signaling pathway is involved in fibrosis and inflammatory responses of myocardial tissue after myocardial infarction (MI). The purpose of our study was to explore the role of SphK1/S1P signaling pathway in myocardial injury after MI. Materials and Methods. We used Sprague-Dawley (SD) rats to make MI models and detected the changes of SphK1 and S1P in rats at 1, 7, and 14 days after MI. SphK1 inhibitor PF543 was used to treat MI rats, and we detected the changes in myocardial function and structure in rats by cardiac function test, 2,3,5-triphenyl tetrazolium staining, and histological staining. In addition, we used H2O2 to induce H9c2 cell injury to investigate the effect of PF543 on the viability of myocardial cells. Results. Myocardial tissue lesions and fibrosis were observed at 7 and 14 days after MI, and the expressions of SphK1 and S1P in the injured myocardial tissues increased significantly in day 7 and day 14 in comparison to the control group. After treatment of MI rats with PF543, the structure of rat myocardial tissue was significantly improved and the degree of fibrosis was reduced. After MI, the expression of α-SMA and collagen I in the myocardium of rats was significantly increased while PF543 decreased their expression. PF543 also improved the cardiac function of MI rats and reduced the expression of IL-1β, IL-6, and TNF-α in the serum. PF543 also increased the viability of H9c2 cells in vitro. Conclusions. The inhibition of the SphK1/S1P signaling pathway contributed to the relief of myocardial injury in MI rats. PF543 improved the myocardial structure and function of MI rats and reduced the level of fibrosis and inflammation in MI rats.