Purple-colored wheat is a kind of wheat with purple-colored grains that contain significant levels of anthocyanins, and anthocyanins benefit human health because of their antioxidant activities. Many studies indicated stresses can significantly induce anthocyanin accumulation during grain development. However, the effects of auxins and cytokinins (CKs) on anthocyanin accumulation during purple grain development have been poorly investigated. Here, we explored the relationships of endogenous auxins and CKs with anthocyanin accumulation during the natural development of purple wheat grains. We found dynamic change of auxin content was consistent with anthocyanin accumulation trend during seed development, which began to rapidly increase from 25 dpa up to the maximum level at 35 dpa. Conversely, CKs, including iPR, cZ, czR, tZ, and tZR, significantly decreased at 25 dpa. The relative expression of GzPIN1, GzLAX3, GzIAA12, GzABP1, GzZOG2, GzUGT, and GzCKX2 was assessed, which is in agreement with ABA and CK accumulation. The findings indicated that auxins and CKs were involved in anthocyanin accumulation during purple grain development in an antagonistic manner. Meanwhile, the contents of the antioxidant enzymes POD and CAT were consistent with the dynamic change in the anthocyanin accumulation rate, which implied that ROS signaling participates in the regulation of anthocyanin accumulation.