Jadomycin B, produced by the soil bacterium Streptomyces venezuelae ISP5230, induces cytotoxicity in human breast cancer cells in vitro and has antitumoral effects in animal models. In models of multidrug-resistant, triple-negative breast cancer, jadomycin B has shown promise as it is not a substrate of ABCB1 and ABCG2 drug efflux transporters. The generation of reactive oxygen species and inhibition of topoisomerases are potential mechanisms of jadomycin B-mediated DNA damage and apoptosis. However, the mechanisms of jadomycin B's anticancer activity have not been fully elucidated. By gradually exposing MDA-MB-231 triple-negative human breast cancer cells to jadomycin B, we hypothesized that resistance could be selected to further understand jadomycin B's pharmacological mechanisms. A 3-fold increase in the jadomycin B IC50 was observed in MDA-MB-231 cells exposed to increasing jadomycin B concentrations (0-3 μM) over 7 months, herein 231-JB cells. The 231-JB cells were cross-resistant to jadomycin F and S but not to the comparator drugs mitoxantrone, doxorubicin, and SN-38. The 231-JB cells did not have increased mRNA expression of topoisomerase-2 nor ABCB1 and ABCG2. Cyclooxygenase-2 (COX-2) increased by 25-fold, but expression of prostaglandin E2 receptor 4 did not significantly change. Cotreatment with celecoxib (15-45 μM), a COX-2 inhibitor, resensitized the 231-JB cells to jadomycin B (IC50 = 1.41 ± 0.24 to 0.75 ± 0.31 μM vs 2.28 ± 0.54 with 0 μM celecoxib). To our knowledge, this work represents the first report of the involvement of COX-2 in jadomycin B activity in vitro, proving to be an exciting new target for the exploration of jadomycin B anticancer activity. SIGNIFICANCE STATEMENT: Cyclooxygenase-2 (COX-2), the rate-limiting enzyme in prostaglandin production, is associated with procancer signaling. COX-2, ABCB1, and ABCG2 overexpression are typically correlated in cancer, contributing to chemotherapy resistance. We observed increased COX-2, but not ABCG2 or ABCB1, expression in 231-JB cells. This indicates that jadomycin B triggers a distinct resistance mechanism. The COX-2 inhibitor celecoxib reversed jadomycin B resistance in 231-JB cells. As such, 231-JB cells represent an important model for COX-2 signaling in breast cancer and jadomycin B mechanism of action.