The aim of this study was to investigate the antiviral effects of salidroside, a major component of Rhodiola rosea L. First, the antiviral effects of salidroside against coxsackievirus B3 (CVB3) were determined in vitro and in vivo. Then, the effect of salidroside on the mRNA expression of some important cytokines was measured in hearts of infected BALB/c mice by RT-PCR. Salidroside exhibited obvious antiviral effects both in in vitro and in vivo experiments. Salidroside was found to modulate the mRNA expression of interferon-gamma (IFN-gamma), interleukin-10 (IL-10), tumor necrosis factor-alpha (TNF-alpha), and interleukin-2 (IL-2). In conclusion, salidroside possesses antiviral activities against CVB3 and it may represent a potential therapeutic agent for viral myocarditis.