Common lambsquarters tolerance to glyphosate is problematic because of the species' widespread distribution, competitive ability with many crop species, the widespread use of glyphosate in agriculture, and the weed's potential to develop decreased sensitivity to multiple herbicide sites of action. The mechanism that confers common lambsquarters tolerance to glyphosate is not known. Therefore, we conducted experiments to determine the mechanism of tolerance to glyphosate in an accession of common lambsquarters from Indiana relative to a sensitive accession from Wisconsin. The ED50 (the effective dose that reduced shoot mass 50% relative to nontreated plants) value for the tolerant accession (1.6 kg ae ha−1 ± 0.4 standard error of the mean [SEM]) was eightfold greater than the ED50 for the sensitive accession (0.2 kg ae ha−1 ± 0.2 SEM) 28 d after treatment. The glyphosate target-site (EPSPS) DNA sequence at proline 106, shikimate accumulation as an estimate of EPSPS sensitivity, and EPSPS protein abundance did not differ between accessions. Absorption of 14C-glyphosate was slightly greater in the tolerant accession than it was in the sensitive accession at 48 and 72 h after treatment (HAT). However, the tolerant accession translocated a smaller percentage of absorbed 14C-glyphosate to the tissue above the treated leaf, which included the shoot apical meristem, at 24, 48, and 72 HAT (P ≤ 0.05, 0.01, and 0.10, respectively). These results suggest an important role of reduced translocation in conferring tolerance of common lambsquarters to glyphosate.