The leaves of Juniperus sabina (Cupressaceae) are used in traditional Uygur medicine for the treatment of rheumatism and arthritic pain. This study aimed to investigate the analgesic and anti-inflammatory effects of total flavonoids from leaves of Juniperus sabina (JSTF) on rodents. The anti-inflammatory activity was investigated using the carrageenan, egg albumin, or histamine-induced rat paw edema as well as xylene-induced ear edema, capillary permeability, and cotton pellet granuloma while the antinociceptive activity was evaluated using the mouse writhing, formalin, and hot-plate tests. JSTF (125, 250, 500 mg/kg) significantly inhibited xylene-induced ear edema in mice (inhibition ratio as 16.22%, 40.67%, and 51.78%, respectively) and also significantly ameliorated acetic acid increased vascular permeability in mice (inhibition ratio as 11.63%, 32.56%, and 53.49%, respectively). JSTF (250 and 500 mg/kg) gave significant reduction of carrageenin-induced paw oedema at the interval of 1 h and 5 h. Administration of JSTF (500 mg/kg) caused a significant anti-inflammatory effect against oedema induced by egg albumin or histamine at the interval of 0.5 h and 4 h, and both which induced the paw oedema were also inhibited by JSTF (250 mg/kg) at a point in 1, 2, or 3 h after the inflammation. Furthermore, JSTF (125, 250, and 500 mg/kg) produced time-course increase in pain threshold in hot-plate test also. JSTF produced concentration-dependent inhibition on cyclooxygenase-2 (COX-2) or 5-lipoxygenase (5-LO) activities in vitro, and their IC50 values were 31.92 and 129.26 μg/mL, respectively. Moreover, JSTF significantly caused a significant dose-dependent inhibition on acetic acid induced writhing response in mice (inhibition ratio as 23.27%, 36.91%, and 50.76%, respectively). JSTF also significantly ameliorated formalin-induced pain in mice in the late phase on dose-dependent way. These results confirms the clinical use of J. sabina for treatment of rheumatoid arthritis in ethnomedicine, and its effective mechanism will be further studied in the future.