This research explored the possible mechanism of Tripterygium wilfordii in suppressing cancer progression. Cancer coli-2 cells were exposed to Tripterygium wilfordii treatment (0, 20, 40, 80 μmol/l) for 24 h. Cell proliferation, clone formation, and apoptosis were monitored using cell counting kit-8 reagent, plate clone formation, and flow cytometry experiments. PRR34-antisense ribonucleic acid 1, miR-3614-5p, and cleavedcaspase-3 contents were assessed using real-time quantitative reverse transcription assay or Western blot. Dual-luciferase reporter system confirmed the targeting between PRR34-antisense ribonucleic acid 1 and miR-3614-5p. After treatment with various doses of Tripterygium wilfordii, cancer coli-2 cell proliferation, inhibition rate, apoptosis rate, cleaved-caspase-3 protein level and miR-3614-5p expression were increased, and clone formation number as well as PRR34-antisense ribonucleic acid 1 expression were decreased in a concentration-dependent manner. PRR34-antisense ribonucleic acid 1 directly targeted miR-3614-5p. Upregulated PRR34-antisense ribonucleic acid 1 partly abolished Tripterygium wilfordii-induced cancer coli-2 cell proliferation repression and apoptosis promotion via targeting miR-3614-5p. Tripterygium wilfordii might hinder cancer coli-2 cell proliferation by regulating PRR34-antisense ribonucleic acid 1/miR-3614-5p.