Objective To investigate the effects of groove topography on morphology and migration speed of cervical cancer HeLa cells. Methods HeLa cells were cultured on PDMS substrates with four different surface features, namely, flat substrate, 10 μm width parallel groove, 20 μm width parallel groove, bifurcate groove. Immunofluorescence technique was used to transfect F-actin in HeLa cells, and specific probes Mito-Tracker Green were used to label mitochondria. The location, morphology of cells and distribution of mitochondrial at different moments were obtained through the living cell system. Results Compared with 20 μm width parallel groove and flat substrate, HeLa cells in 10 μm width parallel groove were more orderly arranged and more elongated, but their migration speed was much slower. HeLa cells at the bifurcation protruded into branches and mitochondria were mainly distributed at the protrusion and around the nucleus. The bifurcation reduced the average migration speed of HeLa cells in 10 μm width parallel groove. Conclusions Groove topography has a significant effect on morphology and migration speed of HeLa cells. The research findings help to understand the role of topography in in vivo microenvironment during migration of HeLa cells, and provide references for the subsequent research on invasion and metastasis of cervical cancers.