The roots of Codonopsis lanceolata (Siebold & Zucc.) Benth. & Hook.f. ex Trautv. have been traditionally used for medicinal purposes across East Asia. However, their chemical constituents in Japanese-grown varieties remain uninvestigated. This study employed ultra-high-performance liquid chromatography–quadrupole–orbitrap mass spectrometry to perform a comprehensive chemical analysis of the roots of C. lanceolata cultivated in Nagano Prefecture, Japan, leveraging fragment pattern analysis of both isolated and commercially available compounds as references compounds. As a result, 27 compounds, including triterpenoid saponins (19–22), polyacetylenes (6, 15, 18), flavonoids (16, 17), phenylpropanoids (3–5, 7, 9), a lignan (10), glycolipids (8, 11–14), phospholipids (23–27), and amino acids (1, 2), were identified. Notably, a triterpenoid saponin (19) was identified as a previously unreported compound, and ten compounds (3, 6, 8, 10, 13, 17, and 23–27) were identified from C. lanceolata roots for the first time. The ex vivo study revealed that lancemaside A (22) exhibited a time-dependent vasodilatory effect on rat aortic ring specimens. These findings not only advanced the understanding of the chemical constituents and biological activity of C. lanceolata roots but also provided valuable insights for their future applications and quality control.