The aim of the present study was to assess the analgesic activity of the macrolide antibiotic tilmicosin at dose levels of 20 and 40 mg/kg of body weight, subcutaneously, against chemical- and thermal-induced acute pains, using acetic acid-induced writhing, formalin-induced pain, hot-plate, and tail-flick models in mice. Tilmicosin showed a dose-dependent significant decrease in the number of writhes in the acetic acid-induced writhing test and significant decrease in hind paw-licking time in the late phase of the formalin test. However, it did not cause any significant changes in the reaction times to heat stimuli in the hot-plate and tail-flick models. In chemically-induced pains, both dose levels of tilmicosin showed significant effects compared to those of the corresponding standard peripheral analgesic, acetylsalicylic acid (200 mg/kg of body weight, subcutaneously) being 26.37 ± 2.88 and 43.64 ± 3.85% vs. 73.35 ± 1.44% in acetic acid test; and 19.23 ± 3.85 and 44.90 ± 1.80% vs. 73.63 ± 2.39% in the late phase of formalin test, respectively. These results may indicate that tilmicosin possesses a significant peripheral but not central analgesic potential that may be beneficial in symptomatic relief of pain when it is used in therapy, in addition to its well-established antibacterial effect.