In the present study, we investigated the role of exogenous long chain ceramide (C16 cer) in transferrin (Tf) internalization and recycling pathways in jurkat human T lymphoma cell line. We traced fluorescently labeled Tf cellular entry, Tf nucleation (Tfn) and recycling and determined the time frame for each step. Clathrin dependent pathway inhibition was carried out by two different means; potassium depletion and chlorpromazine (CPZ) to investigate the entry pathway after treatment with C16 cer. The level of transferrin receptor (TfR), clathrin and cleaved caspase 3 were carried out by Western blot. There were obvious differences between control and C16 cer treated cells in the kinetics of Tf up taking and recycling. Formation of Tfn in C16 cer treated cells was different in time, size and shape compared with control cells. Also, results indicated that C16 cer changed the Tf internalization pathway to become clathrin independent pathway. This mis-internalization of Tf leads to loss of cell viability and finally apoptotic cell death. The current obtained data may be applicable for clathrin dependent machinery used by different receptors and agents.