Drug resistance exists as a major obstacle in the treatment of cancer, and drug molecules that retain effectiveness against resistant cancers are a high clinical priority. Ethyl 2-amino-6-(3,5-dimethoxyphenyl)-4-(2-ethoxy-2-oxoethyl)-4H-chromene-3-carboxylate (CXL017) was recently identified as a promising lead for the treatment of multidrug-resistant leukemia, which elicits its cytotoxic effect, in part, through inhibition of the sarco/endoplasmic reticulum Ca(2+)-ATPase (SERCA). Herein initial experiments with SERCA1a and CXL017 demonstrated no significant effect on calcium affinity, competed with ATP, and induced a dose-dependent decrease in ATPase activity. Among all CXLs tested, (-)-CXL017 exhibited the greatest SERCA inhibition with an IC50 = 13.5 ± 0.5 μM. Inhibitor combination studies were used to assess potential interactions between (-)-CXL017 and well-known SERCA inhibitors: thapsigargin, cyclopiazonic acid, and 2,5-di-tert-butylhydroquinone. Surprisingly, (-)-CXL017 exhibited marked synergy with each of the known SERCA inhibitors, whereas all combinations of the known inhibitors yielded additive effects, indicating that (-)-CXL017 may bind at a unique allosteric site. Treatment of parental (HL60) and multidrug-resistant (HL60/MX2) acute myeloid leukemia cells with the known SERCA inhibitors revealed that all of these inhibitors demonstrate selective cytotoxicity (7.7-400-fold) for the resistant cell line. Within the CXL series, a positive correlation exists between SERCA inhibition and cytotoxicity in HL60/MX2 but not HL60. (-)-CXL017 was also shown to enhance the cytotoxicity of thapsigargin in HL60/MX2 cells. Given the elevated SERCA levels and ER calcium content in HL60/MX2, SERCA likely plays a significant role in the collateral sensitivity of this multidrug-resistance cell line to CXL molecules as well as known SERCA inhibitors.