Lung cancer, similar to other chronic diseases, occurs due to perturbations in multiple signaling pathways. Mono-targeted therapies are not ideal since they are not likely to be effective for the treatment and prevention of lung cancer, and are often associated with drug resistance. Therefore, the development of multi-targeted agents is required for novel lung cancer therapies. Thioredoxin reductase (TrxR or TXNRD1) is a pivotal component of the thioredoxin (Trx) system. Various types of tumor cells are able to overexpress TrxR/Trx proteins in order to maintain tumor survival, and this overexpression has been shown to be associated with clinical outcomes, including irradiation and drug resistance. Emerging evidence has indicated that oleanolic acid (OA) and its derivatives exhibit potent anticancer activity, and are able to overcome drug resistance in cancer cell lines. In the present study, it was demonstrated that a novel synthesized OA family compound, olean-28,13b-olide 2 (OLO-2), synergistically enhanced cisplatin (CDDP)-mediated apoptosis, led to the activation of caspase-3 and the generation of reactive oxygen species (ROS), induced DNA damage, and inhibited the activation of the extracellular-signal-regulated kinase (ERK), signal transducer and activator of transcription 3 (STAT3), AKT and nuclear factor-κB (NF-κB) pathways in human multidrug-resistant A549/CDDP lung adenocarcinoma cells. Subsequent analyses revealed that OLO-2 inhibited P-glycoprotein (P-gp or ABCB1) and TrxR by reducing their expression at the protein and mRNA levels, and by suppressing P-gp ATPase and TrxR activities. Further biological evaluation indicated that OLO-2 significantly reduced Trx and excision repair cross-complementary1 (ERCC1) protein expression and significantly inhibited the proliferation of drug-sensitive (A549) and multidrug-resistant (A549/CDDP) non-small cell lung cancer (NSCLC) cells, but had no effect on non-tumor lung epithelial-like cells. In addition, the present study demonstrated, for the first time, to the best of our knowledge, that overexpressing or knocking down TrxR in NSCLC cells enhanced or attenuated, respectively, the resistance of NSCLC cells against CDDP, which indicated that TrxR plays an important role in CDDP resistance and functions as a protector of NSCLC against chemotherapeutic drugs. OLO-2 treatment also exhibited up to 4.6-fold selectivity against human lung adenocarcinoma cells. Taken together, the results of the present study shed light on the drug resistance-reversing effects of OLO-2 in lung cancer cells.