This work reports the design and use of a gradient nanotopography to enhance the resistance of mammalian cell adhesion on surfaces. The gradient nanotopography is prepared by depositing gold films on a glass microscope slide in a single azimuthal direction, but with continuous increase in the angle between the incidence of gold atoms and the normal of the surface. By forming patterned self-assembled monolayers (SAMs) of alkanethiols into cell-adhesive regions (HS(CH2)(14)CH3) surrounded by cell-resistant areas (HS(CH2)(12)(OCH2CH2)(3)OH) on the gold film, the adhesion and confinement of albino 3T3 fibroblasts along the topographic gradient are studied. At the early stage of cell culture, cells adhere faster and reach confluency sooner on higher topographic regions (gold deposited at large incident angle) than on lower topographic regions (gold deposited at small incident angle). After cells proliferate to reach confluency within the patterns, the cells are confined in the high topographic regions up to 25 days, which is about 4 times longer than on gold films prepared on a rotating planetary. Overall, introducing nanostructured topography in gold films significantly enhances the bioinert chemistry of tri(ethylene glycol)-terminated SAMs that resist the adhesion of mammalian cells.