Natural halloysite nanotubes (HNTs), with nanotubular structure, are attracting considerable attention in recent years. The hollow tubular structure allows HNTs to play an important role in drug delivery system as drug carriers. However, the wide applications of HNTs in biomedicine have been hampered by the lack of sufficient intracellular researches so far. In this study, we systemically investigated the transport mechanisms of HNTs in A549 living cells. The colocalization and inhibition experiments illustrated FITC-labeled HNTs were readily internalized into cells by both clathrin- and caveolae-dependent endocytosis, and the transport pathway of HNTs is an actin- and microtubule-associated process via Golgi apparatus and lysosome. Meanwhile, the cell cycle assay clarified that HNTs can prompt the intracellular transportation of gemcitabine and enhance the gemcitabine concentration in A549 tumor cells. Such elucidation of intracellular transport pathway of HNTs offers insights into the site-specific delivery and cellular internalization of HNTs, which provide a reasonable guidance for the design of novel drug delivery system.