The association between women's stress and pregnancy glucose levels remain unclear, specifically when considering the preconception period as a sensitive window of exposure. We investigated whether preconception perceived stress was associated with glucose levels during pregnancy among women attending a fertility center (2004-2019). Before conception, women completed a psychological stress survey using the short version of the validated Perceived Stress Scale 4 (PSS-4), and blood glucose was measured using a 50-gram glucose load test during late pregnancy as a part of screening for gestational diabetes. Linear and log-binomial regression models were used to assess associations of total PSS-4 scores with mean glucose levels and abnormal glucose levels ( ≥ 140 mg/dL), adjusting for age, body mass index, race, smoking, education, physical activity, primary infertility diagnosis, number of babies, and mode of conception. Psychological stress was positively associated with mean abnormal glucose levels. The adjusted marginal means (95% CI) of mean glucose levels for women in the first, second, and third tertiles of psychological stress were 115 (110, 119), 119 (115, 123), and 124 (119, 128), and mg/dL, respectively (P for trend = .007). Also, women in the second and third tertiles of psychological stress had 4% and 13% higher probabilities of having abnormal glucose compared with women in the first tertile of psychological stress (P trend = .01). These results highlight the importance of considering preconception when evaluating the relationship between women's stress and pregnancy glucose levels.