Previous studies showed that the cytoplasmic transport of nanoparticles to the nucleus is driven by a vesicular sorting system. Artificial approaches for targeting a microtubule-associating motor complex is also a challenge. We describe herein the development of a liposomal nanoparticle, the surface of which is modified with stearylated octa-arginine (STR-R8), and a dynein light chain (LC8)-associated peptide derived from an African swine fever virus protein p54 (p54(149-161)) with polyethyleneglycol (PEG) as a spacer (p54(149-161)-PEG/R8-liposomal nanoparticles (LNPs)). The p54(149-161)-PEG/R8-LNPs preferentially gain access to the nucleus, resulting in a one- to two-order of magnitude higher transfection activity in comparison with p54(149-161)-free nanoparticles (PEG/R8-LNPs). Further studies of particle tracking in HeLa cells stably expressing green fluorescent protein (GFP)-tagged tubulin (GFP/Tub-HeLa) indicate that p54(149-161) stimulated the transport of nanoparticles along fibrous tubulin structures. Moreover, a part of the p54(149-161)-PEG/R8-LNPs appeared to undergo quasi-straight transport without sharing the tracks corresponding to PKH67, the plasma membrane of which had been prestained with a marker just before transfection, while corresponding movement was never observed in the case of PEG/R8-LNPs. These findings suggest that a portion of the p54(149-161)-modified nanoparticles can use microtubule-dependent transport without the need for an assist by a vesicular sorting system.