Human LINE-1 retrotransposon encodes two proteins (ORF1p and ORF2p), which are required for LINE-1 retrotransposition. However, since the detail mechanism of ORF1p regulation remains elucidation, this mass spectrometry data set provides a comprehensive list of ORF1p interacting proteins in HeLa cells and will contribute to more understanding of LINE-1 retrotransposition and host defense mechanisms.