Micropillar compression was used to investigate whether Ag segregation to an asymmetric Σ5001 grain boundary will lead to measurable strength differences compared to the pure copper bicrystal. Ag segregation was accomplished by deposition and subsequent annealing of an Ag thin-film applied on the surface of the Cu bicrystal. Atom probe tomography analysis indicated Ag segregation at the grain boundary with a peak concentration of 2.3 at.%. While the pristine Σ5 grain boundary shows a yield strength of 288 ± 18 MPa when compressing 1 µm diameter pillars along 〈001〉, micropillars containing an Ag-segregated Σ5 grain boundary demonstrated an increased yield strength of 318 ± 17 MPa. In addition, post-deformation electron microscopy was carried out to examine the active slip systems and slip transmission across Ag-free and Ag-containing bicrystals. The results are compared to reference measurements of the adjacent single crystal grains. The 1 µm pillar diameter promoted deformation governed by dislocation-grain boundary interactions for the bicrystalline pillars. This is the first time that changes in flow stress associated with grain boundary segregation have been quantified locally without interference from other mechanisms such as solid solution strengthening, formation of precipitates or changes in stacking fault energy. The results clearly indicate that purely geometrical models for slip transmission are not sufficient as the local atomic structure and composition influence dislocation transmission through grain boundaries. Display omitted