S-amlodipine has been broadly used to treat hypertension, but its protective effects and underlying mechanism remain controversial. The purpose of our study was to investigate the mechanism by which S-amlodipine improves endothelial dysfunction. Specifically, we investigated if S-amlodipine regulates RANK/RANKL/OPG and micro-RNA 155 (miR-155) levels. Spontaneous hypertensive rats (SHR) were randomly divided into two groups: SHR (n = 12) and S-amlodipine (n = 12). We found that left ventricular ejection fraction (LVEF) increased significantly in the S-amlodipine group compared to the SHR group. After 10 weeks of S-amlodipine treatment, tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) levels were significantly lower and eNOS and NO production was significantly higher in the S-amlodipine group compared to the SHR group. In human umbilical vein endothelial cells (HUVECs), miR-155, RANK, and RANKL levels were significantly decreased, while OPG mRNA levels were significantly increased in the S-amlodipine group. HUVECs were transfected with miR-155 mimics or an inhibitor to determine the relationship between miR-155 and RANK/RANKL/OPG and NF-κB signaling. OPG mRNA levels following miR-155 inhibition were significantly higher compared to levels following treatment with miR-155 mimics. S-amlodipine significantly inhibited RANKL expression and NF-κB phosphorylation, and there were no significant differences in response to the NF-κB inhibitor (Bay110785). RANKL expression and NF-κB phosphorylation significantly decreased in the miR-155 inhibitor group. Furthermore, OPG protein expression significantly increased in response to miR-155 inhibition and S-amlodipine treatment (all p < 0.05). Our results indicate that S-amlodipine inhibits inflammation and protects against endothelial dysfunction, likely via regulating the RANK/RANKL/OPG pathway, which appears to be downstream of miR-155.