Since the combination of anticancer drugs and opioids is very common, apatinib and tramadol are likely to be used in combination clinically. Based on this, it is particularly important to explore the effect of apatinib on the metabolism of tramadol. This study evaluated the effects of apatinib on the pharmacokinetics of tramadol and its main metabolite o-desmethyltramadol in SD rats and the inhibitory effects of apatinib on tramadol in rat liver microsomes (RLMs), human liver microsomes (HLMs) and recombinant human CYP2D6.1. The samples were determined by ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS). The in vivo results showed that compared with the control group, apatinib increased the AUC(0-t), AUC(0-∞) and Cmax values of tramadol and O-desmethyltramadol, and decreased the values of VZ/F and CLz/F. In addition, the MRT(0-t), MRT(0-∞) values of O-desmethyltramadol were increased. In vitro, apatinib inhibited the metabolism of tramadol by a mixed way with IC50 of 1.927µM in RLMs, 2.039µM in HLMs and 15.32µM in CYP2D6.1.