Liposomes are widely recognized as effective drug delivery systems, characterized by biodegradability, biocompatibility, and ability to minimize toxicity. However, liposome-based nanotechnology has not demonstrated superior anti-tumor efficacy due to their limited intratumor penetration. Strategies to improve the tumor delivery efficiency of nanomedicine remain to be developed. Moreover, the specific steps involving inter-/intra-cellular pathways in delivery could not be fully revealed by real experiment. Mathematical modeling is a great choice. Hence, this study analyzed the roles of physicochemical properties of liposomes and tumorassociated environment in intratumoral penetration, using ten anti-tumor liposomes datasets to develop two kinetic models in tumor spheroid cells based on transcytosis and paracellular transport mechanisms. Modeling results reveal the dominated penetration pathway of liposomes studied through the paracellular pathway compared to transcytosis. Liposomes with positive surface charge and high membrane fluidity enhance the maximal binding capacity on the cell membrane. Smaller liposome sizes promote internalization on the cell membrane, leading to increased drug accumulation within the cell. The pattern of liposome penetration remained consistent across different tumor-associated environments. Our developed kinetic models accurately described the penetration process of liposomes in multicellular tumor spheroid, offering valuable insights for the development of new nano antitumor medications with similar characteristics from a pharmacokinetic perspective at the tissue level.