Conducting probe atomic force microscopy (CP-AFM) was used to examine electrical transport through an individual grain boundary (GB) in the organic semiconductor sexithiophene (6T, Egap ∼ 2.3 eV). The sample consisted of a pair of grains grown by vapor deposition onto an SiO2/Si substrate. A variable channel length transistor was constructed using a microfabricated Au electrode contacting one grain, a Au-coated AFM tip as a positionable electrode, and the doped Si substrate as a gate. The GB resistance was found to be gate voltage dependent and large, on the order of 109-1010 Ω for a 1 μm boundary length. Resistances across single 6T grains were an order of magnitude lower. The results indicate that GBs can be the principal bottleneck to charge transport in polycrystalline organic semiconductor films, particularly at low gate fields, consistent with a recent model that proposes potential barriers exist between grains. We estimate the GB barrier height to be on the order of 100 meV.