Objective To study the effect of cordycepin on invasion and metastasis of Hela cervical cancer cells by regulating miR-135b-5p expression and its regulatory mechanism. Methods Hela cells were treated with 50 and 100 μmol/ L cordycepin, and Hela cell growth was measured by MTT assays. Transwell assays were used to assess Hela cell invasion, a scratch assay was used to assess the metastasis ability of Hela cells, and Real-time PCR was used to measure the miR-135-5p expression level in human immortalized epidermal cell line Hacat and Hela cells, and the effect of various cordycepin concentrations on miR-135-5p expression level. miR-135b-5p mimic was transfected into Hela cells, and the invasion and metastasis of Hela cells were assessed by Transwell and scratch assays. Results After cordycepin treatment, Hela cell growth was significantly inhibited, and the inhibitory effect of the high concentration cordycepin was more obvious(P<0. 05). The miR-135b-5p expression level in Hela cells was (1. 97±0. 07), which was significantly higher than that in Hacat cells(1. 01±0. 03), and the difference between groups was statistically significant(t=28. 187, P=0. 000). After cordycepin treatment, the invasion and metastasis rates of Hela cells and miR-135b-5p expression were decreased significantly. After treatment with a high cordycepin concentration, the invasion and metastasis rates of Hela cells were lower, and the differences between groups were statistically significant(t= 138. 614~317. 100, P<0. 05). Overexpression of miR-135b-5p significantly increased the invasion and metastasis rates of Hela cells(t= 7. 145, 7. 465, P<0. 05), and vimentin expression was increased and E-cad expression was decreased(t= 8. 223, 7. 473, P< 0. 05). Conclusions Cordycepin inhibits the invasion and metastasis of Hela cells by inhibiting miR-135b-5p expression.