Objective This study aimed to investigate the effect of betamethasone treatment on the endocervical concentration of IL-1β, IL-4, IL-6, and TNF-α in preterm labor patients. Study design We studied 68 prime-gravid women in preterm labor between 34 and 37 weeks of gestation without clinical infection. Endocervical concentrations of inflammatory cytokines were assessed; immediately on admission and 48 h after administration of two doses of intramuscular betamethasone (12 mg/kg). Wilcoxon and Mann–Whitney tests along with χ 2 and Student's t tests were utilized for statistical analysis. Results In the betamethasone group IL-1β and TNF-α significantly decreased ( P < 0.001), and IL-6 and IL-4 increased ( P: NS). Among patients delivered before or on the 7th day of admission IL-6 and TNF-α were higher at the most significant levels ( P < 0.001) compared to IL-1β and IL-4 ( P: 0.001, 0.002 in respect). Conclusion Betamethasone can help induce the down regulation of endocervical inflammatory cytokines in patients with preterm labor.