Display omitted •Caffeine promoted growth and migration of paclitaxel-treated cancer cells.•Caffeine inhibited apoptosis in paclitaxel-treated cancer cells.•Caffeine down-regulated paclitaxel-induced α-tubulin acetylation in vitro and in vivo.•Caffeine decreased paclitaxel-induced inhibition of tumor growth. Caffeine (1,3,7-trimethylxanthine) is a xanthine alkaloid found in a number of dietary products consumed worldwide, such as coffee, tea, and soft beverages, and is known to act as a modifying agent for cytotoxic chemotherapeutic drugs. Studies have shown that caffeine reduces the cytotoxic effects of paclitaxel and inhibits paclitaxel-induced apoptosis; however, the underlying mechanism remains unclear. Here, we investigated whether caffeine inhibits the antitumor activity of paclitaxel via down-regulation of α-tubulin acetylation. In vitro studies, involving MTT assay, wound-healing assay, cell apoptosis assay, and western blotting analysis of A549 and HeLa cells, were performed. A549 and HeLa cell-based xenografts were established, and western blotting and immunohistochemical staining were performed for in vivo studies. The results showed that caffeine promoted the growth of cancer cells treated with paclitaxel. Additionally, caffeine enhanced migration ability, inhibited apoptosis, and decreased the acetylation of α-tubulin in paclitaxel-treated cancer cells. Furthermore, caffeine decreased the inhibitory effect of paclitaxel on tumor growth through down-regulation of α-tubulin acetylation in vivo. Taken together, these findings demonstrate that caffeine inhibits the anticancer activity of paclitaxel via down-regulation of α-tubulin acetylation, suggesting that patients receiving treatment with taxanes, such as paclitaxel, should avoid consuming caffeinated beverages or foods.