Rationale Studies identified kininogen as a potential biomarker of preeclampsia, a major cause of adverse maternal outcomes. High‐molecular‐weight kininogen (HK) and its activated form participate in numerous pathways associated with establishing and maintaining pregnancy. However, dynamic changes in HK and naturally occurring HK‐derived peptides during the natural course of pregnancy are largely unknown. Methods Longitudinal serum samples during the course of normal pregnancy (trimesters T1, T2, T3) from 60 pregnant women were analyzed by western blot with an anti‐HK antibody. Circulating peptides in longitudinal serum specimens derived from 50 participants were enriched using nanoporous silica thin films. Peptides were identified by liquid chromatography/tandem mass spectrometry (LC/MS/MS) and database searching. Relative quantification was performed using MaxQuant and in‐house scripts. Normality was evaluated by either ANOVA or Friedman tests with p < 0.05 for statistical significance. Results Western blotting revealed that HK significantly decreased during normal pregnancy (T1 vs T2, p < 0.05; T1 vs T3, p < 0.0001). A 100 kDa intermediate increased during pregnancy (T1 vs T2, p < 0.005; T1 vs T3, p < 0.01). Moreover, the heavy chain (T1 vs T2, p < 0.0001; T1 vs T3, p < 0.0001; T2 vs T3, p < 0.01) and light chain (T1 vs T2, p < 0.0001; T1 vs T3, p < 0.0001; T2 vs T3, p < 0.05) significantly increased during pregnancy. LC/MS/MS analysis identified 180 kininogen‐1 peptides, of which 167 mapped to domain 5 (D5). Seventy‐three peptides with ten or more complete data sets were included for further analysis. Seventy peptides mapped to D5, and 3, 24, and 43 peptides showed significant decrease, no trend, and significant increase, respectively, during pregnancy. Conclusions This study demonstrates dynamic changes in HK and naturally occurring HK‐derived peptides during pregnancy. Our study sheds light on the gestational changes of HK and its peptides for further validation of them as potential biomarkers for pregnancy‐related complications.