Ceramide (CE)-based combination therapy (CE combination) as a novel therapeutic strategy has attracted great attention in the field of anti-cancer therapy. The principal purposes of this study were to investigate the synergistic effect of CE in combination with docetaxel (DTX) (CE + DTX) and to explore the synergy mechanisms of CE + DTX. The 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and combination index (CI) assay showed that simultaneous administration of CE and DTX with a molar ratio of 0.5:1 could generate the optimal synergistic effect on murine malignant melanoma cell (B16, CI = 0.31) and human breast carcinoma cell (MCF-7, CI = 0.48). The apoptosis, cell cycle, and cytoskeleton destruction study demonstrated that CE could target and destruct the microfilament actin, subsequently activate Caspase-3 and induce apoptosis. Meanwhile, DTX could target and disrupt the microtubules cytoskeleton, leading to a high proportion of cancer cells in G2/M-phase arrest. Moreover, CE plus DTX could cause a synergistic destruction of cytoskeleton, which resulted in a significantly higher apoptosis and a significantly higher arrest in G2/M arrest comparing with either agent alone (p < 0.01). The in vivo antitumor study evaluated in B16 tumor-bearing mice also validated the synergistic effects. All these results suggested that CE could enhance the antitumor activity of DTX in a synergistic manner, which suggest promising application prospects of CE + DTX combination treatment.