The contribution of second messenger signaling, glucose level and sex hormones to sexual dimorphism in the streptozotocin model of diabetic painful peripheral neuropathy was evaluated. Streptozotocin induced elevation of blood glucose and mechanical hyperalgesia (measured by the Randall-Selitto paw-withdrawal test) were both greater in female rats. Ovariectomy abolished and estrogen implants reconstituted this sexual dimorphism; gonadectomy in males had no effect. An inhibitor of protein kinase Cϵ attenuated hyperalgesia in males and ovariectomized females, but not in normal females or in ovariectomized females with estrogen implants, whereas inhibitors of protein kinase Cδ attenuated hyperalgesia in females but not in males. Inhibitors of protein kinase A, protein kinase C (non-selective), protein kinase G and nitric oxide synthase attenuated hyperalgesia equally in both sexes. Higher blood glucose levels in diabetic females were also sex hormone dependent, and magnitude of hyperalgesia correlated with blood glucose level in diabetic male and female rats. These results demonstrate sexual dimorphism in diabetic hyperalgesia, mediated by sex hormone dependent differences in protein kinase Cϵ and protein kinase Cδ signaling and blood glucose levels and suggest that sex may be an important factor to be considered in the treatment of symptomatic diabetic neuropathy.