Scanning transmission electron microscope (STEM) through-focus imaging (TFI) has been used to determine the three-dimensional atomic structure of Bi segregation-induced brittle Cu grain boundaries (GBs). With TFI, it is possible to observe single Bi atom distributions along Cu  twist GBs using an aberration-corrected STEM operating at 200 kV. The depth resolution is ~5 nm. Specimens with GBs intentionally inclined with respect to the microscope’s optic axis were used to investigate Bi segregant atom distributions along and through the Cu GB. It was found that Bi atoms exist at most once per Cu unit cell along the GB, meaning that no continuous GB film is present. Therefore, the reduced fracture toughness of this particular Bi-doped Cu boundary would not be caused by fracture of Bi–Bi bonds.