Gemcitabine is widely used for anticancer therapy. However, its short blood circulation time and poor stability greatly impair its application. To solve this problem, we prepared a poly (l-glutamic acid)-g-methoxy poly (ethylene glycol)-gemcitabine conjugate (l-Gem) with a 14.3 wt% drug-loading content. l-Gem showed concentration- and time-dependent cytotoxicity towards 4T1, LLC, MIA PaCa-2 and A2780 in vitro. Pharmacokinetic and biodistribution studies indicated that l-Gem had remarkably enhanced blood stability, prolonged blood circulation time and greatly improved selective tumor distribution compared with free gemcitabine. The area under the concentration–time curve from zero to infinity [AUC(0–∞)] of l-Gem in plasma was 43-fold higher than that of free gemcitabine. The AUC(0–∞) of the inactive metabolite, 2′-deoxy-2′,2′-difluorouridine in the l-Gem group was ˜20% of that observed in the free gemcitabine group. The drug tumor accumulation ratio in the l-Gem group relative to the free gemcitabine group was 9.9 at 36 h, while the tumor AUC ratio was 15.8. Testing on Balb/C mice bearing the 4T1 tumor further demonstrated that l-Gem had significantly higher anticancer efficacy than free gemcitabine in vivo. These findings indicated that l-Gem has great potential for cancer treatment.