The human hepatitis B virus X protein (HBx) is known as a dual-specificity transactivator stimulating the transcriptional machinery in the nucleus and signal transduction pathways in the cytoplasm. HBx-induced activation of mitogen-activated protein kinase (MAPK) signaling cascades is considered to play an important role in hepatitis B virus-mediated hepatocarcinogenesis. Herein, we have identified the regions of HBx that are crucial for activating such signaling cascades in vivo. A truncated mutant incorporating regions C–E (amino acids 58–140) was as effective as the full-length HBx in activating MAPKs and enhancing activator protein-1 binding activity. While deletion of region C (amino acids 58–84) or D (amino acids 85–119) led to a drastic loss of function, region E (amino acids 120–140) was dispensable for the activation of signaling cascades. Overall, these findings provide the first evidence for the requirement of domain 58–119 of HBx in transmitting mitogenic signals to the nucleus in vivo.