Abstract: The Tanlu fault zone is the largest and most active Quaternary fault in eastern China. An M-8.5 earthquake occurred in 1668 in Tancheng City located on the southern segment of the Tanlu fault. In this study, we analyzed the crustal velocity structure of the Tanlu fault zone and assessed its relationship with the regional seismic activity, using a large dataset of the arrival times of the primary (P) and secondary (S) waves recorded at 121 permanent seismic stations in and around the middle-southern segments of the Tanlu fault; in total, we used the data of 8528 local earthquakes. We developed three-dimensional (3D) images of the P-wave (Vp) and S-wave (Vs) velocities and the Vp/Vs ratio, using the TOMOG3D model (a seismic tomography model). The tomography results revealed the following features: (1) a significant velocity segmentation feature exists in the middle-southern segments of the Tanlu fault zone; this feature can be divided into Section A (located in the north of Tancheng), Section B (extending from Tancheng to Mingguang), and Section C (extending from Mingguang to Wuxue). (2) Large earthquakes are mainly distributed at the junction of the high- and low-velocity anomalies. (3) A strong correlation was noted between the velocity structure and seismic activity, especially in Suqian (where a clear low-velocity anomaly that corresponded to a seismic gap was observed). Our study provides new insights into the crustal heterogeneity and seismic activity in the middle-southern segments of the Tanlu fault zone.