The toxicity of pesticides to organisms depends on the total amount of chemical exposure. Toxicity can be minimized if the organism recognizes the pesticide and alters its behavior. Furthermore, the physical barrier of cuticular hydrocarbons can prevent the entrance of the pesticide into the organism. Finally, if the pesticide enters the body, the organism experiences physiological changes favoring detoxification and the maintenance of homeostasis. We analyzed the behavioral and metabolic response of the spider Polybetes pythagoricus at different times of exposure to the organophosphate pesticide chlorpyrifos. First we observed that the individuals are capable of recognizing and avoiding surfaces treated with pesticides based on a behavioral analysis. Subsequently, we characterized cuticular hydrocarbons as a possible barrier against pesticides. Then we observed that the pesticide provoked histological damage, mainly at the level of the midgut diverticula. Finally, we analyzed the activity of several of the spider's enzymes linked to oxidative stress after exposure to chlorpyrifos for different lengths of time (6, 24, and 48 h). We observed that catalase activity was high at the start, whereas the activity of superoxide dismutase and glutathione S-transferase changed significantly at 48 h. Lipid peroxidation became high at 6 h, but decreased at 48 h. In conclusion, although P. pythagoricus can avoid contact with chlorpyrifos, this pesticide causes activation of the antioxidant system when it enters the body. Our results make a significant contribution to the ecotoxicology of spiders.