Most cells, highly sensitive to oxygen levels, undergo apoptosis under hypoxia. Therefore, the involvement of hypoxia in rotator cuff tendon degeneration has been proposed. While previous studies have reported that hypoxia induces apoptosis in rotator cuff fibroblasts (RCFs), little research has investigated whether antioxidants have cytoprotective effects against RCF apoptosis. The present study aimed at determining whether the antioxidant N-acetylcysteine (NAC) exerted cytoprotective effects against hypoxia-induced RCF apoptosis. Third-passage rat RCFs were divided into normoxia, NAC, hypoxia and NAC-hypoxia groups. The hypoxia inducer was 1,000 µmol/L cobalt chloride (CoCl2); the antioxidant was 20 mmol/L NAC. Expressions of hypoxia-inducible factor-1α (HIF-1α) and heme oxygenase-1 (HO-1), cell viability, intracellular reactive oxygen species (ROS) production, apoptosis rates as well as expressions of cleaved caspase-3, cleaved poly ADP-ribose polymerase-1 (PARP-1), vascular endothelial growth factors-β (VEGF-β) and matrix metalloproteinase-2 (MMP-2) were evaluated. Expression of HIF-1α and HO-1 was significantly higher in the hypoxia group than in the normoxia group (p < 0.001). Cell viability was significantly lower in the hypoxia group than in the normoxia group (p < 0.001). Intracellular ROS production, apoptosis rate and expressions of cleaved caspase-3, cleaved PARP-1, VEGF-β and MMP-2 were significantly higher in the hypoxia group than in the normoxia group (p < 0.001). All these responses were significantly attenuated by pre-treatment with NAC (p ≤ 0.001). ROS were involved in hypoxic RCF apoptosis induced by CoCl2; NAC, an ROS scavenger, inhibited hypoxia-induced RCF apoptosis by inhibiting ROS production.