In this study, we investigated the influence of paclitaxel nanocrystals (PTX-NC) surface charge on cell internalization and cell cytotoxicity. PTX-NCs were prepared using the nano-precipitation method. The surface-modified PTX-NCs were prepared using an absorption method with positively charged poly(allylamine hydrochloride) (PAH) and negatively charged poly(sodium 4-styrenesulfonate) (PSS). The morphologies of the surface-modified and unmodified PTX-NCs were characterized by field emission scanning electron microscopy. An in vitro drug release study was performed in phosphate-buffered saline (pH 7.4) containing 0.5% (w/v) Tween 80 for 48 h. Cell internalization was evaluated at time intervals of 0.5, 1, and 2 h, and cell cytotoxicity was analyzed for 24 h using A549 cells. Three different types of PTX-NCs with a mean size of around 300-400 nm were successfully prepared. The zeta-potential revealed PSS-PTX-NCs (-22.7 ± 5.1 mV), PTX-NCs (− 2.4 ± 2.9 mV), and PAH-PTX-NCs (+ 19.3 ± 3.4 mV). The three types of PTX-NC exhibited higher drug release than pure PTX. The positive charge on PTX-NC resulted in higher cell uptake and cell cytotoxicity than the negative charge on PTX-NC. Moreover, the positive charge on PTX-NC showed stronger interactions with bovine serum albumin. In conclusion, the positive charge on PTX-NCs improved cell internalization, cell cytotoxicity, and interactions with bovine serum albumin.