Targeting tumor metabolic vulnerabilities such as "glutamine addiction" has become an attractive approach for the discovery of novel antitumor agents. Among various mechanisms explored, SLC1A5, a membrane transporter that plays an important role in glutamine cellular uptake, represents a viable target to interfere with tumor's ability to acquire critical nutrients during proliferation. In the present study, a stably transfected HEK293 cell line with human SLC1A5 (HEK293-SLC1A5) was established for the screening and identification of small molecule SLC1A5 inhibitors. This in vitro system, in conjunction with direct measurement of SLC1A5-mediated L-glutamine-2,3,3,4,4-D5 (substrate) uptake, was practical and efficient in ensuring the specificity of SLC1A5 inhibition. Among a group of diverse compounds tested, mianserin (a tetracyclic antidepressant) demonstrated a marked inhibition of SLC1A5-mediated glutamine uptake. Subsequent investigations using SW480 cells demonstrated that mianserin was capable of inhibiting SW480 tumor growth both in vitro and in vivo, and the in vivo antitumor efficacy was correlated to the reduction of glutamine concentrations in tumor tissues. Computational analysis revealed that hydrophobic interactions between SLC1A5 and its inhibitors could be a critical factor in drug design. Taken together, the current findings confirmed the feasibility of targeting SLC1A5-mediated glutamine uptake as a novel approach for antitumor intervention. It is anticipated that structural insights obtained based on homology modeling would lead to the discovery of more potent and specific SLC1A5 inhibitors for clinical development.