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The rapid transmission of vaccinia virus (VACV) in vivo is thought to be closely related to the cell migration induced by it. Cell migration involved in dynamic changes of cell-substrate adhesion and actin cytoskeleton organization, which can influence by the micro/nano-scale topographic structures that cells are naturally exposed to via contact guidance. However, migration behaviors of VACV-infected cells exposed to topographic cues are still unknown. Herein, we designed an open chip with microgrooved poly(dimethyl siloxane) (PDMS) substrate to explore the topography roles in VACV-induced cell migration. Differed from the random cell migration observed in traditional scratch assay on planar substrate, VACV-infected cells had a tendency to persistently migrate along the axis parallel to microgroove with increased velocity. Moreover, infected cells exhibited a dominant elongated protrusion aligned to the micro-grating axis compare to the shorter lamella extended in any direction on smooth substrate. Interestingly, the Golgi complex preferred to relocate behind the nucleus confined within the micro-grating axis in majority of infected migratory cells. The directional polarization of cells embodied in protrusion formation and Golgi reorientation was responsible for the directionally persistent migration behaviors induced by VACV on microgrooved substrate. Infected cells response to substrate topography, causing the actin-filled stretched protrusion containing numerous virions and accelerated movement is likely to facilitate direct and rapid spread of VACV. This work opens a window for us to understand the migration behaviors of infected cells in vivo, and also provides a cue for revealing the relationship between virus-induced cell migration and virus rapid spread.