Spontaneous preterm birth (PTB) is a major obstetrical problem around the globe and the mechanisms leading to PTB are unclear. Recently, changes in the circulating levels of placental extracellular vesicles (EVs) during pregnancy have been associated with various pregnancy complications. However, progress in the field is hindered by the inability to isolate placental EVs from the maternal circulation. A longitudinal study design was used to determine the protein cargo present in circulating placental EVs in maternal plasma of term and PTB across gestation (ie, first, second, and third trimester). Placental-derived EVs were enriched from the total EV population based on their expression of membrane-bound placental alkaline phosphatase (PLAP). A quantitative, information-independent acquisition (sequential windowed acquisition of all theoretical mass spectra [SWATH]) approach identified and quantified the placental EV protein contents. PLAP(+) EVs did not change in characteristics (size, shape, and markers) but did differ in numbers across gestation with low levels in PTB. A comparison analysis between the PLAP(+) EV proteome from term and PTB revealed 96 proteins differing significantly (P < 0.05, false discovery rate 1%) across gestation. Bioinformatics analysis of differentially expressed proteins revealed consistent upregulation of inflammatory pathways in both upregulation of epithelial mesenchymal transition pathways at term and downregulation of coagulation/complement activation in preterm. Characterization of the proteomic profile in PLAP(+) EVs across gestation demonstrates dramatic changes, which might be used to understand the biological process associated with early parturition and develop biomarkers for predicting high-risk status for PTB.