Spring viraemia of carp virus (SVCV) is the causative pathogen of the outbreaks of an acute haemorrhagic and contagious viraemia responsible for the significant mortality in several cyprinid species. However, the endocytic pathway(s) and their regulatory molecules have not been characterized for SVCV. Here, using a combination of specific pharmacological inhibitors, transmission electron microscopy, immunofluorescence microscopy and real-time quantitative PCR, we found that SVCV entered grass carp ovary cells via clathrin-mediated endocytosis and macropinocytosis in a low-pH-dependent manner. We also discovered that dynamin II, actin microfilaments and microtubules were essential for SVCV internalization. Moreover, we found that the P21-activated kinase 1 inhibitor IPA-3 and the protein kinase C inhibitor rottlerin could block SVCV cell entry and replication, while phosphatidylinositol 3-kinase inhibitors wortmannin and LY294002 could promote SVCV infection. Results presented in this study provide helpful insight into revealing the initial steps of SVCV infection, and they may facilitate the development of therapeutic interventions.