Human antigen (Hu) R is an RNA-binding protein whose overexpression in human cancer correlates with aggressive disease, drug resistance, and poor prognosis. HuR inhibition has profound anticancer activity. Pharmacologic inhibitors can overcome the limitations of genetic inhibition. In this study, we examined the antitumor activity of CMLD-2, a small-molecule inhibitor directed against HuR, using non-small cell lung cancer (NSCLC) as a model. CMLD-2 efficacy was tested in vitro using H1299, A549, HCC827, and H1975 NSCLC cells and MRC-9 and CCD-16 normal human fibroblasts. Treatment of NSCLC cells with CMLD-2 produced dose-dependent cytotoxicity, caused a G1 phase cell-cycle arrest and induced apoptosis. CMLD-2 decreased HuR mRNA and the mRNAs of HuR-regulated proteins (Bcl2 and p27) in tumor cells. Additionally, reduction in the expression of HuR, Bcl2, cyclin E, and Bcl-XL with increased expression of Bax and p27 in CMLD-2-treated NSCLC cells were observed. CMLD-2-treated normal cells, HuR-regulated mRNAs and proteins albeit showed some reduction were less compared to tumor cells. Finally, CMLD-2 treatment resulted in greater mitochondrial perturbation, activation of caspase-9 and -3 and cleavage of PARP in tumor cells compared to normal cells. Our proof-of concept study results demonstrate CMLD-2 represents a promising HuR-targeted therapeutic class that with further development could lead to advanced preclinical studied and ultimately for lung cancer treatment.