Purpose The primary aim of this study was to determine cabazitaxel’s affinity for the ABCB1 /P-glycoprotein (P-gp) transporter compared to first-generation taxanes. Methods We determined the kinetics of drug accumulation and retention using 14 C-labeled taxanes in multidrug-resistant (MDR) cells. In addition, membrane-enriched fractions isolated from doxorubicin-selected MES-SA/Dx5 cells were used to determine sodium orthovanadate-sensitive ATPase stimulation after exposure to taxanes. Custom 3 H-azido-taxane analogues were synthesized for the photoaffinity labeling of P-gp. Results The maximum intracellular drug concentration was achieved faster with 14 C-cabazitaxel (5 min) than 14 C-docetaxel (15–30 min). MDR cells accumulated twice as much cabazitaxel than docetaxel, and these levels could be restored to parental levels in the presence of the P-gp inhibitor PSC-833 (valspodar). Efflux in drug-free medium confirmed that MDR cells retained twice as much cabazitaxel than docetaxel. There was a strong association ( r 2  = 0.91) between the degree of taxane resistance conferred by P-gp expression and the accumulation differences observed with the two taxanes. One cell model expressing low levels of P-gp was not cross-resistant to cabazitaxel while demonstrating modest resistance to docetaxel. Furthermore, there was a 1.9 × reduction in sodium orthovanadate-sensitive ATPase stimulation resulting from treatment with cabazitaxel compared to docetaxel. We calculated a dissociation constant (Kd) value of 1.7 µM for 3 H-azido-docetaxel and ~ 7.5 µM for 3 H-azido-cabazitaxel resulting in a 4.4 × difference in P-gp labeling, and cold docetaxel was a more effective competitor than cabazitaxel. Conclusion Our studies confirm that cabazitaxel is more active in ABCB1 (+) cell models due to its reduced affinity for P-gp compared to docetaxel.