SummarySf9 cells (a clonal isolate of Spodoptera frugiperda Sf21 cells) are commonly used to generate recombinant virus‐like particles (VLPs). For VLPs generation, Sf9 cells are infected with recombinant baculoviruses (rBV) expressing desired proteins. During rBV infections, Sf9 cells have changes in cell diameters and surface structures. In this study, for the first time, we investigated nanostructural changes of Sf9 cells using atomic force microscopy (AFM) during VLPs generation containing Toxoplasma gondii rhoptry protein 18 (ROP 18). As results, Sf9 cells were changed to be larger at 2 days after rBV infections. They maintained their sizes and morphologies on day 3 and 4. Based on morphological (perimeter and diameter) and surface roughness (roughness average and root mean square) changes of Sf9 cells observed by AFM, we inferred that these nanostructural changes in Sf9 cell membranes might be due to the production and extrusion of VLPs after rBV infection. Our results suggest that shape and roughness parameters of Sf9 cell morphology and membrane surface by AFM could be very effective for quantitative analysis of VLP production. This study provides important information about structural and mechanochemical properties of Sf9 cells which are closely related with biological function. SCANNING 38:735–742, 2016. © 2016 Wiley Periodicals, Inc.