Problem The immune system represents a leading pathway of interest in the pathophysiology of preterm birth. The majority of human clinical studies interrogating this pathway have utilized circulating immune biomarkers; however, these concentrations typically reflect only basal production but not key functional properties of the immune system, particularly variation in the pro‐inflammatory response to antigen challenge and the regulation of this response. Thus, in this study, we utilized an ex vivo stimulation protocol that quantifies these processes, and we examined their prospective association with the gestation length and risk of preterm birth. Method of Study Immune responsiveness and regulation were assessed in 128 pregnant women in mid‐gestation using an ex vivo stimulation protocol. Maternal pro‐inflammatory responsivity of leukocytes was quantified by assessing the release of the pro‐inflammatory cytokines IL‐6, TNF‐α, and IL‐1β in response to antigen stimulation, and regulation of the pro‐inflammatory response was quantified by assessing the suppression of stimulated cytokine response upon co‐incubation with increasing dexamethasone concentrations (ie, glucocorticoid receptor resistance; GRR). Results Higher maternal GRR, indicating impaired regulation of the pro‐inflammatory response, was significantly and independently associated with shorter gestational length (β = −0.42, p = .0091) and a 3.0‐fold increase in risk for preterm birth (OR = 3.01, 95% CI = 1.17–7.70, p = .0218). Basal circulating IL‐6 and TNF‐α were not associated with either outcome. Conclusion The association of maternal GRR with length of gestation and preterm birth risk suggests that the processes represented by this measure—maternal pro‐inflammatory propensity and immune regulation—may provide further mechanistic insight into the pathophysiology of preterm birth.