Background <p>Serine/arginine-rich splicing factor 1 (SR1), an orthologue of hypothetical RNA-binding protein (HRB1) in yeast, is essential to the asexual development of <i>Plasmodium falciparum</i> and <i>P. berghei</i>. However, their interacting proteins in malaria parasites remain unclear.</p> Methods <p>To identify SR1-interacting proteins in malaria parasites, transgenic <i>Pb</i> ANKA expressing green fluorescent protein-fused PbSR1 (PBANKA_1232100) was generated and performed immunoprecipitation coupled to mass spectrometry (IP-MS) using the transgenic parasites. To investigate the developmental stage at which PbSR1 and RhopH2 are expressed, transgenic parasites co-expressing the fusion proteins PbSR1::GFP and RhopH2::mCherry were generated and western blot analysis and live-cell fluorescence imaging were performed.</p> Results <p>The fluorescence signal of PbSR1::GFP was stronger in nuclei than the cytoplasm of malaria parasites. IP-MS of the transgenic parasites indicated that PbSR1 interacts with nuclear proteins, including RNA-binding protein and small nuclear ribonucleoprotein, and cytoplasmic proteins, such as RhopH1 (or Clag3), RhopH2, and RhopH3. Live-cell fluorescence imaging showed that co-localization of PbSR1 and RhopH2 in the cytoplasm was observed in trophozoites and gametocytes but not mature schizonts and merozoites. From these results, trophozoites, immature schizonts and gametocytes are candidate stages at which cytoplasmic PbSR1 interacts with RhopH2.</p> Conclusions <p>PbSR1 interacts with nuclear proteins and rhoptry proteins.</p>