Objective To investigate the cause of tumor cell migration by comparing the effect of substrate stiffness on hepatic and hepatoma carcinoma cell migration so as to understand the invasive characteristics of tumor cells. Methods Immunofluorescence staining, morphological analysis and transwell were employed to observe the morphological characteristics of HCCLM3 and L02 cells on different substrates and test their migration characteristics with the quantitative analysis. Results (1) The migration rate and net translocation of HCCLM3 and L02 cells on 4 kPa substrate was higher than those both on 0.5 kPa(most soft one) and on glass (the hardest one) substrates, and L02 cells also displayed higher migration efficiency than HCCLM3 cells on such substrates. (2) The mean squared displacement of HCCLM3 and L02 cells on different substrates showed consistent tendency, and the directional persistence of L02 cells on the softer substrate was significantly higher than that of HCCLM3 cells. (3) In 0.5 and 1 mg/mL three dimensional collagen environment, the number of invasive cells of HCCLM3 was remarkably more than that of L02 cells. After adding MMPs inhibitor GM6001 (40 μg/mL), the number of invasive cells was notably increased in HCCLM3 cells, but notably decreased in L02 cells. Conclusions (1) In two dimensional comparatively soft environment, L02 cells displayed an efficient migration due to its higher directional persistence. (2) In three-dimensional collagen environment, the invasion efficiency of HCCLM3 cells was significantly higher due to the various modes of migration adaptation to the microenvironment.