Light Detection and Ranging (LiDAR) remote sensing technology provides a more efficient means to acquire accurate 3D information from large-scale environments. Among the variety of LiDAR sensors, Multi-Beam LiDAR (MBL) sensors are one of the most extensively applied scanner types for mobile applications. Despite the efficiency of these sensors, their observation accuracy is relatively low for effective use in mobile mapping applications, which require measurements at a higher level of accuracy. In addition, measurement instability of MBL demonstrates that frequent re-calibration is necessary to maintain a high level of accuracy. Therefore, frequent in situ calibration prior to data acquisition is an essential step in order to meet the accuracy-level requirements and to implement these scanners for precise mobile applications. In this study, kinematic in situ self-calibration of a backpack-based MBL system was investigated to develop an accurate backpack-based mobile mapping system. First, simulated datasets were generated for the experiments and tested in a controlled environment to inspect the minimum network configuration for self-calibration. For this purpose, our own-developed simulator program was first utilized to generate simulation datasets with various observation settings, network configurations, test sites, and targets. Afterwards, self-calibration was carried out using the simulation datasets. Second, real datasets were captured in a kinematic situation so as to compare the calibration results with the simulation experiments. The results demonstrate that the kinematic self-calibration of the backpack-based MBL system could improve the point cloud accuracy with Root Mean Square Error (RMSE) of planar misclosure up to 81%. Conclusively, in situ self-calibration of the backpack-based MBL system can be performed using on-site datasets, reaching the higher accuracy of point cloud. In addition, this method, by performing automatic calibration using the scan data, has the potential to be adapted to on-line re-calibration.