BACKGROUND Ceramides are known to show anti-cancer activity. A novel ceramide analog, (S,E)-3-hydroxy-2-(2-hydroxybenzylidene)amino-N-tetradecylpropanamide (analog 315) was developed as part of a larger study focused on finding more effective breast cancer treatments. OBJECTIVE To assess whether analog 315 shows any or a combination of the following effects in breast cancer cells in vitro: inhibiting proliferation, inducing apoptosis, and altering protein expression. Also, to determine whether it inhibits chemo-resistant breast cancer tumor growth in vivo mouse model. METHODS In vitro cell proliferation and apoptosis after treatment with analog 315 were assessed in three breast cancer cell lines (MCF-7, MCF-7TN-R, and MDA-MB-231) and reported. Protein expression was assessed by microarray assay. For the in vivo studies, chemo-resistant breast cancer cells were used for tumor development in two groups of mice (treated and control). Analog 315 (25 mg/kg/day) or control (dimethyl sulfoxide) was administered intraperitoneally for 7 days. Effects of analog 315 on inhibiting the growth of chemo-resistant breast cancer tumors after treatment are reported. RESULTS Analog 315 reduced MCF-7TN-R chemo-resistant tumor burden (volume and weight) in mice. Liver metastasis was observed in control mice, but not in the treated animals. Ki-67, a proliferation marker for breast cancer cells, increased significantly ( P  < 0.05) in control tumor tissue. In vitro studies showed that analog 315 inhibited cell proliferation, altered protein expression and induced apoptosis in all three breast cancer cell lines studied, of which the effects on MCF-7TN-R cells were the most significant. CONCLUSIONS Analog 315 reduced tumor growth in chemo-resistant breast cancer, inhibited cell proliferation, altered protein expression, and induced apoptosis in all three cell lines studied.