A two-dimensional column-switching system without sample loop trapping, where two columns were switched directly via a six-port two-position switching valve, was successfully applied for the first time to the isolation and purification of six iridoid glycosides including geniposide, gardenoside, shanzhiside, scandoside methyl ester, deacetyl-asperulosidic acid methyl ester and genipin-1-beta-D-gentiobioside from Gardenia jasminoides Ellis, a plant used in the traditional Chinese medicine. The introduction of the six-port switching valve instead of sample loop assured 100% recovery from the first dimension to the second, and the injection volumes of the second dimension could reach 20 ml. In this mode of operation, the sample size of the two-dimensional approach was more than 1.3 times that of conventional gradient methods with even less solvent consumption. And the simultaneous operations of the two dimensions allowed the cycle time to be less than 19 min, compared with that (90 min) in the gradient elution single-dimension mode of operation. All of the six isolated iridoid glycosides were isolated at high purities of over 99% with approximately 96% recoveries.