This study aimed to investigate the effect of pyrethroid insecticides on the regulation of bovine cervical function. Cervical cells or strips obtained from cows during the periovulation period were treated with cypermethrin and fenvalerate (0.1–10 ng/mL). None of the pyrethroids exerted a cytotoxic effect, whereas only fenvalerate increased the cervical contraction force and mRNA expression of receptor of oxytocin and prostaglandin (PG) synthases. Both pyrethroids inhibited PG secretion and decreased the amount of diacylglycerol, which is the second messenger involved in oxytocin signal transmission, and fenvalerate decreased the myosin light-chain kinase level. These findings indicate that fenvalerate induces greater disruption of cervical function than cypermethrin.