Cancer cells highly express amino-acid transporters in order to sustain their rapid metabolism and growth. ASCT2 is one of the primary glutamine transporters expressed in various cancers. Several researches have shown that ASCT2 inhibitors and disruption of ASCT2 gene suppress cell proliferation and tumor growth. Here, we generated a novel anti-human ASCT2 rat monoclonal antibody (mAb) designated as Ab3-8, and investigated whether the Ab3-8 had anti-cancer effects on KRAS mutant cancer cells. Rats were immunized with RH7777 rat hepatoma cells stably expressing green fluorescent protein (GFP)-fused human ASCT2 proteins. Splenocytes from these rats were fused with X63 mouse myeloma cells and Ab3-8 was selected from hybridomas by flow cytometry. Ab3-8 reacted with various human cancer cell lines, and inhibited intracellular glutamine uptake and phosphorylation of AKT and ERK in SW1116 and HCT116 human colorectal cancer cells with KRAS-mutation. Furthermore, in vivo xenograft study has shown that Ab3-8 suppress tumor growth derived from these cells and phosphorylation of AKT and ERK in the tumor. These results suggest that ASCT2-targeted cancer therapy is a promising strategy for KRAS-mutated cancers.