A series of novel thioether andrographolide derivatives were synthesized by incorporating various aromatic (or heteroaromatic) substituents into C-12 or 14-OH. A total of 38 andrographolide derivatives were prepared and evaluated for their <i>in vitro</i> inhibitory activity against cancer cells. All the derivatives exhibited better activity against prostate cancer cells (PC-3) than the parent compound. Among these, compounds <b>6a</b>, <b>8</b>, <b>9</b>, <b>17</b>, <b>19</b>, <b>31</b>, and <b>32</b> demonstrated good activity. These compounds were further evaluated for their anticancer activities against other cancer cell lines including MCF-7, MDA-MB-231, and A549. Compounds <b>31</b> and <b>32</b> showed excellent activity against MCF-7 with an IC<sub>50</sub> value of 0.7 and 0.6 μM, respectively. The absolute configuration of <b>15a</b> was determined <i>via</i> single-crystal X-ray diffraction. The activity of <b>6a</b> (12<i>S</i>), which was the precursor of <b>15a</b>, was better than that of the diastereoisomer <b>6b</b> (12<i>R</i>). Moreover, the preliminary structure-activity relationship has been summarized. The results obtained herein are very important for further optimization of andrographolide.