One of the important criteria for achieving efficient nanoparticle-based targeted drug delivery is that the drug is not prematurely released at off-target sites. Here we report the preclinical evaluation of a serum-stable dendrimer-based drug conjugate capable of actively targeting into prostate cancer (PC) cells, delivered through the prostate-specific membrane antigen (PSMA). Multiple molecules of PSMA-binding small molecule glutamate urea (GLA; targeting agent) and the drug methotrexate (MTX) were conjugated to generation S PAMAM dendrimer (G5) through Cu-free "click" chemistry. The GLA was conjugated through a stable amide bond, and the MTX was conjugated either through ester (Es)- or amide (Am)-coupling, to generate G5-GLA(m)-(Es)MTXn and G5-GLA(m)-(Am)MTXn, respectively. In serum-containing medium, free MTX was slowly released from "G5-GLA(m)-(Es)MTXn", with similar to 8% MTX released from the dendrimer in 72 h, whereas the MTX on G5-GLA(m)-(Am)MTXn was completely stable. The G5-GLA(m)-(Am)MTXn bound and internalized into PSMA-expressing LNCaP cells, but not into PSMA-negative PC3 cells. The conjugate-inhibited recombinant dihydrofolate reductase and induced potent cytotoxicity in the LNCaP cells, but not in the PC3 cells. Similar to the action of free GLA, stable amide-linked dendrimer-GLA was capable of inhibiting the enzyme N-acetylated alpha-linked acidic dipeptidase (NAALADase) activity of PSMA. The G5-GLA(m)-MTXn may serve as a serum-stable nanoparticle conjugate to specifically and effectively target and treat PSMA-overexpressing prostate tumors.