Aim To investigate the role and mechanism of Ras homolog gene family member A (RhoA)/Rho-associated coiled-coil forming protein kinase (ROCK) pathway in hypoxia/reoxygenation (H/R)-induced injury in human cardiac AC16 cells. Methods AC16 cells cultured in vitro were divided into control group (normal culture), H/R group (construction of H/R model), H/R＋NC-siRNA group (H/R model was constructed after transfection of NC-siRNA) and H/R＋RhoA-siRNA group (H/R model was established after transfection of RhoA-siRNA), MTT assay was used to detect the survival rate of AC16 cells, the apoptosis rate of AC16 cells was detected by flow cytometry, the activities of lactate dehydrogenase (LDH) and Caspase-3 in AC16 cells were detected by colorimetry, the levels of interleukin-6 (IL-6), interleukin-1β (IL-1β) and tumor necrosis factor-α (TNF-α) in the supernatant of AC16 cells were detected by ELISA, the levels of superoxide dismutase (SOD) and malondialdehyde (MDA) in AC16 cells were detected by kit, the mRNA expression levels of RhoA, ROCK1, ROCK2, nuclear factor-κB (NF-κB) p65 and IKB kinase (IKK) in AC16 cells were detected by real-time fluorescence quantitative PCR, the protein expression levels of RhoA, ROCK1, ROCK2, NF-κB p65, phosphorylated NF-κB (p-NF-κB) p65 and IKK in AC16 cells were detected by Western blot. Results Compared with those in the control group, the cell survival rate and SOD level in H/R group were significantly lower, apoptosis rate, LDH activity, Caspase-3 activity, MDA level, IL-6, IL-1β and TNF-α levels in cell supernatant, RhoA, ROCK1, ROCK2, NF-κB p65, p-NF-κB p65, IKK mRNA and protein expression levels were significantly higher (P＜0.05). There was no significant difference in the above indexes between H/R＋NC-siRNA group and H/R group (P＞0.05). Compared with those in H/R＋NC-siRNA group, the cell survival rate and SOD level in H/R＋RhoA-siRNA group were significantly higher, apoptosis rate, LDH activity, Caspase-3 activity, MDA level, IL-6, IL-1β and TNF-α levels in cell supernatant, RhoA, ROCK1, ROCK2, NF-κB p65, p-NF-κB p65, IKK mRNA and protein expression levels were significantly lower (P＜0.05). Conclusion Targeted inhibition of RhoA/ROCK pathway can reduce H/R-induced cardiomyocyte injury by reducing inflammatory response, oxidative stress and apoptosis.