Expressed on human tumors, the GM3(Neu5Gc) ganglioside represents a promising target for cancer immunotherapy. In absence of functional CMAH in humans, an enzyme required for Neu5Gc sialic acid biosynthesis, tumor-associated Neu5Gc expression has been linked to dietary uptake and metabolic incorporation. The use of cell lines for testing GM3(Neu5Gc)-targeting strategies is complicated by the complexity of sialic acid biology and the influence of culture conditions. Here, quantitative flow cytometry revealed absence of surface GM3(Neu5Gc) in several human cell lines and also mouse cell lines with low CMAH expression. Hypoxia-induced expression of this ganglioside on human SKOV3 cells was observed upon culture in Neu5Gc-containing medium without evidence for CMAH-independent biosynthesis. Mouse Cmah genetic transfection induced stable GM3(Neu5Gc) surface expression in human and mouse cell lines, resulting in effective antitumor responses by the GM3(Neu5Gc)-targeting humanized antibody 14F7hT in vitro and in vivo. This is the first description of the antitumor activity of 14F7hT against human cancer cells. These results contribute to validate GM3(Neu5Gc) as a relevant target for cancer immunotherapy and reinforces the value of 14F7hT as a novel therapeutic tool.