Hepatitis B virus X protein (HBx) possesses a BH3-like motif that directly interacts with the anti-apoptotic proteins, Bcl-2 and Bcl-x. Here we report the interaction between the HBx BH3-like motif and Bcl-x, as revealed by nuclear magnetic resonance spectroscopy. Our results showed that this motif binds to the common BH3-binding hydrophobic groove on the surface of Bcl-x, with a binding affinity of 89 μM. Furthermore, we examined the role of the tryptophan residue (Trp120) in this motif in Bcl-x binding using three mutants. The W120A mutant showed weaker binding affinity (294 μM) to Bcl-x, whereas the W120L and W120F mutants exhibited almost equivalent binding affinity to the wild-type. These results indicate that the bulky hydrophobic residues are important for Bcl-x binding. The findings will be helpful in understanding the apoptosis networks between viral proteins and host factors.