Background: Emerging evidence suggests that biological intermediates play an important role in initiating fine particulate matter (PM_2.5)-associated prohypertensive pathways, but sensitive biomarkers for this pathway are lacking. Aim: To explore whether short-term exposure to PM_2.5 is associated with the concentration of 20-hydroxyeicosatetraenoic acid (20-HETE), a potent vasoactive lipid relevant to the pathophysiology of hypertension. Methods: In this longitudinal panel study, we repeatedly (up to seven times) measured the blood concentrations of 20-HETE in 120 adults living in Beijing, China. Ambient exposure metrics included the concentrations of hourly PM_2.5 mass and daily PM_2.5 constituents, including three carbonaceous components, eight water-soluble ions, and 16 trace elements. Linear mixed-effects models were used to examine the associations between the change in the 20-HETE concentration and short-term exposure to ambient PM_2.5 metrics after adjustment for age, sex, body mass index, behavioral exposure, socioeconomic characteristics, and meteorological factors. Results: The interquartile range (IQR) increase in the 7–15-hour-lag exposure to PM_2.5 (80 μg/m³) was associated significantly with a 5.3% (95% confidence interval [CI], 0.1–10.7%) to 6.5% (95% CI, 1.7–11.6%) increase in the blood concentration of 20-HETE. The magnitude of the association differed by age, sex, prediabetic status, obesity, and hypertensive status, with a significantly greater increase in 20-HETE observed among those with fasting plasma glucose concentrations ≥ 6.1 mmol/L. In addition to the PM_2.5 mass, the 20-HETE concentration was associated consistently with IQR increases in the 1-day lag exposure to organic carbon (5.7%), black carbon (9.5%), nitrate (3.9%), chloride (2.9%), copper (5.5%), zinc (4.7%), barium (4.1%), and lead (6.2%). The organic carbon estimate was robust in the two-pollutant models. Furthermore, increased 20-HETE correlated with elevated blood pressure (BP), although no mediation of 20-HETE on PM_2.5-associated BP change was found. Conclusions: The 20-HETE blood concentration increased significantly in response to short-term exposure to ambient PM_2.5, which may be partly responsible for the prohypertensive effects of PM_2.5.