Objective. To investigate the effects of berberine (Berb) on dexamethasone- (Dex-) induced injury of human tendon cells and its potential mechanism. Methods. CCK-8 assay was used to explore the appropriate concentration of Dex-induced injury of tendon cells and the doses of Berb attenuates Dex cytotoxicity; cell wound healing assay was used to detect the effects P < 0.05 of Berb and Dex on the migration ability of tendon cells; flow cytometry was used to measure cell apoptosis; DCF DA fluorescent probe was used to measure the ROS activity of cells. Western blotting was used to detect the expression of phenotype related factors including smooth muscle actin α (SMA-α), type I collagen (Col I), col III, apoptosis-related factors, caspase-3, cleaved caspase-3, caspase-9, cleaved caspase-9, and PI3K/AKT. Results. CCK-8 assay showed that 1–100 μM Dex significantly inhibited the proliferation of tendon cells in a concentration-dependent manner P < 0.05 , where the inhibitory effect of 100 μM Dex was most significant P < 0.005 , and the pretreatment of 150, 200 μM Berb could reverse those inhibitions (all P < 0.05). Compared with the control group, Dex significantly inhibited cell migration P < 0.05 , while Berb pretreatment could enhance cell migration P < 0.05 . Flow cytometry and ROS assay showed that Dex could induce apoptosis and oxidative stress response of tendon cells (all P < 0.05), while Berb could reverse those responses P < 0.05 . Western blot showed that Dex could inhibit the expression of the col I and III as well as α-SMA (all P < 0.05) and enhance the expression of apoptosis-related factors including cleaved caspase-3 and cleaved caspase-9 (all P < 0.05). Besides, Dex could also inhibit the activation of the PI3K/AKT signaling pathway (all P < 0.05), thus affecting cell function, while Berb treatment significantly reversed the expression of those above proteins (all P < 0.05). Conclusion. Berb attenuated DEX induced reduction of proliferation and migration, oxidative stress, and apoptosis of tendon cells by activating the PI3K/AKT signaling pathway and regulated the expression of phenotype related biomarkers in tendon cells. However, further studies are still needed to clarify the protective effects of Berb in vivo.