Thermal creep Knudsen pumps are made up of micro/nanochannels subjected to a temperature gradient. In such a combination, the fluid flows from the cold side to the hot side based on the thermal creep phenomenon. Due to the widespread use of Knudsen pumps in various applications, much research has been done to improve their technology. In this work, the effects of wall surface roughness on the flow field characteristics are investigated using the molecular-based direct simulation Monte Carlo method. The surface roughness is modeled by square bumps on the wall. The roughness parameters, including relative roughness, roughness aspect ratio, and roughness distance, are studied in a wide range, that is, 0 ≤ ε ≤ 10, 0.5 ≤ ξ ≤ 2, and 3 ≤ χ ≤ 10, respectively. It is concluded that the existence of roughness, no matter how small, has a significant effect on the flow conditions; so that the subminiature roughness of ε = 0.5% results in a 24% decrease in the mass flow rate. This emphasizes the importance of considering the role of surface roughness in the calculation of the performance of Knudsen pumps. Furthermore, the results indicate that the mass flow rate is independent of the relative roughness and the roughness distance for ε ≤ 1.25% and χ ≤ 5, respectively.