One new secoiridoid glycoside with conjugated diene, named 3-epi-swertiajaposide C (1), was isolated from the whole plants of Gentiana apiata N.E. Br., together with 11 known compounds, 7-deoxyloganic acid (2), isoorientin (3), gentiopicroside (4), silybin B (5), swertiamarin (6), asystasioside A (7), 6'-O-beta-D-glucopyranosylgentiopicroside (8), umbelliferone (9), oleanolic acid (10), kaempferol (11), and beta-sitosterol (12). The structure of the new compound (1) was elucidated on the basis of spectroscopic evidence including UV, IR, MS, NMR, HMBC, HMQC, and NOESY. Compounds 1, 2, 5, 6, 8, 9, and 11 were found in this plant for the first time. Moreover, silybin B (5) was isolated from the other plants besides Silybum marianum (L.) Gaertn for the first time by the present study.