Field studies were conducted in commercial muscadine vineyards in western North Carolina in 2018 and eastern North Carolina in 2019, 2020, and 2021 to determine tolerance of younger (< 9 yr) and older (≥ 9 yr) bearing muscadine grapevines to 2,4-D postemergence (POST)-directed beneath the crop. Treatments included 2,4-D choline at 0, 0.53, 1.06, 1.60, and 2.13 kg ae ha-1 applied as a single treatment in May or June (spring) at immediate pre-bloom, and sequential treatments at 0.53 followed by (fb) 0.53, 1.06 fb 1.06, 1.6 fb 1.6, or 2.13 fb 2.13 kg ha-1. The first application of the sequential treatments was applied in spring fb another application of the same amount in July (summer) at pre-veraison. No differences were observed for injury on muscadine grapevines from 2,4-D treatments. Differences among treatments were not observed for yield of younger vines. However, with respect to yield of older vines, a difference due to 2,4-D rate was observed in 2018 where yield was higher for 2,4-D at 1.6 kg ha-1 compared to the nontreated, and 2,4-D at 0.53 and 2.13 kg ha-1. A rate by timing interaction was observed in 2019 with lower yield from 0.53 kg ha-1 2,4-D summer application compared to all other summer treatments but similar to the nontreated. However, no biological pattern was observed from either of these differences. No differences among treatments were observed for fruit pH, titratable acidity (TA), or soluble solid content (SSC) of either younger or older vines.