Celastrol has been identified as a potential candidate for anticancer drug development. In this study, 28 novel celastrol derivatives with C‐6 sulfhydryl substitution and 20‐substitution were designed and synthesized, and their antiproliferative activity against human cancer cells and non‐malignant human cells was evaluated, with cisplatin and celastrol being used as controls. The results showed that most of the derivatives had enhanced in vitro anticancer activity compared to the parent compound celastrol. Specifically, derivative 2f demonstrated the most potent inhibitory potential and selectivity against HOS with an IC50 value of 0.82 μM. Our study provides new insights into the structure–activity relationship of celastrol and suggests that compound 2f may be a promising drug candidate for the treatment of osteosarcoma. In this study, 26 novel celastrol analogues with C‐6 sulfhydryl substituted were designed, synthesized, and evaluated their antiproliferative activity against human cancer cells (HCT116, A549, HOS, 5‐8F, and M231). What's more, this study gives a new insight into the structure–activity relationships of celastrol.