OBJECTIVE： To study in vitro anticancer activity of conjugated linoleic acid-gemcitabine conjugate （CLA-GEM）. METHODS： IC50 of different tumor cells （breast cancer MCF-7 cell， breast cancer MDA-MB-231 cell， lung cancer A549 cell， small cell lung cancer NCI-H446 cell， glioma C6 cell） were investigated after treated with different concentrations （0.001-100        μmol/L） of CLA-GEM and gemcitabine （GEM） for 72 h； survival rates of MCF-7 cell were investigated after treated with above solution for 24， 48 and 72 h. The dependence of 0.001-100 μmol/L CLA-GEM and GEM to nucleoside transporter was investigated （by IC50） through MCF-7 cells and MDA-MB-231 cells treated with nucleoside transporter inhibitors （NBMPR， 100 μmol/L） and dipyridamole （4 μg/ml）. The change of MCF-7 cell cycle was investigated after treated with 1 μmol/L CLA-GEM and GEM for 24 h. RESULTS： Compared with GEM， IC50 of MCF-7， MDA-MB-231 and NCI-H446 cells became lower after treated with CLA- GEM （P＜0.01）， there were no statistical significances in IC50 between A549 and C6 cells （P＞0.05）. Survival rate of MCF-7 cells decreased significantly after treated with GEM for 48 h and CLA-GEM for 24 h. Survival rate of MCF-7 cells was the lowest， being 21％ after treated with GEM for 72 h， while tumor cells were sacrificed by CLA-GEM completely. Compared with GEM or CLA-GEM， IC50 of MCF-7 and MDA-MB-231 cells increased significantly after treated with NBMPR， dipyridamole combined with GEM （P＜0.01）； there were no statistical significance in IC50 after treated with NBMPR， dipyridamole combined with CLA-GEM （P＞0.05）. Compared with GEM， CLA-GEM could prolong 6％ of S stage of MCF-7 cells （P＜0.01）. CONCLUSIONS： Compared with GEM， CLA-GEM exhibits significant antitumor activity and rapid action， and it isn’t influenced by nucleic acid transportation.