Tendinopathy is one of the most common musculoskeletal disorders with significant repercussions on quality of life and sport activities. Physical exercise (PE) is considered the first-line approach to treat tendinopathy due renowned mechanobiological effects on tenocytes. Irisin, a recently identified myokine released during PE, has been recognized for several beneficial effects towards muscle, cartilage, bone, and intervertebral disc tissues. The aim of this study was to evaluate the effects of irisin on human primary tenocytes (hTCs) in vitro. Human tendons were harvested from specimens of patients undergoing anterior cruciate ligament reconstruction (n = 4). After isolation and expansion, hTCs were treated with RPMI medium (negative control), interleukin (IL)−1β or tumor necrosis factor-α (TNF-α) (positive controls;​ 10 ng/mL), irisin (5, 10, 25 ng/mL), IL-1β or TNF-α pretreatment and subsequent co-treatment with irisin, pretreatment with irisin and subsequent co-treatment with IL-1β or TNF-α. hTC metabolic activity, proliferation, and nitrite production were evaluated. Detection of unphosphorylated and phosphorylated p38 and ERK was performed. Tissue samples were analyzed by histology and immunohistochemistry to evaluate irisin αVβ5 receptor expression. Irisin significantly increased hTC proliferation and metabolic activity, while reducing the production of nitrites both before and after the addition of IL-1β and TNF-α. Interestingly, irisin reduced p-p38 and pERK levels in inflamed hTCs. The αVβ5 receptor was uniformly expressed on hTC plasma membranes, supporting the potential binding of irisin. This is the first study reporting the capacity of irisin to target hTCs and modulating their response to inflammatory stresses, possibly orchestrating a biological crosstalk between the muscle and tendon.