In this manuscript, the effects of Mo content on the microstructure and impact toughness of X80 thick-walled low-temperature pipeline steel were studied. Two test steels with different Mo content (0.25% and 0.40%) were prepared by the thermo-mechanical control process. The impact properties were measured at −45 °C, and the microstructure evolution was observed via an optical microscope (OM), a scanning electron microscope (SEM), electron back-scattered diffraction (EBSD), and a transmission electron microscope (TEM). Each steel showed the formation of a mixed microstructure consisting of polygonal ferrite (PF), granular bainite (GB), and lath bainite (LB). Increasing Mo content resulted in the rise of LB at the expense of PF and GB. At the same time, the morphology of martensite/austenite (M/A) constituents changed from blocky to slender. The dislocation density in the ferrite matrix around the M/A constituents enhanced with an increase in Mo content. This also led to an increase in the microstrains around the M/A constituents. Also, the number fraction of the high angle grain boundary (HAGB) (MTA > 15°) decreased with the addition of more Mo content. Furthermore, with an increase in Mo content from 0.25% to 0.40%, the low-temperature impact toughness decreased from 206 to 57 J. Both an increase in the slender M/A constituents and a decrease in the HAGB number fraction deteriorated the low-temperature impact toughness of the X80 thick-walled low-temperature pipeline steel.