Streptozocin (STZ)-induced diabetic rats show hyperalgesia that is partially attributed to altered protein kinase C (PKC) activity. Both attenuated neuronal nitric oxide synthase (nNOS)-cGMP system and tetrodotoxin-resistant (TTX-R) Na channels in dorsal root ganglion neurons may be involved in diabetic hyperalgesia. We examined whether PKCbeta inhibition ameliorates diabetic hyperalgesia and, if so, whether the effect is obtained through action on neurons by testing nociceptive threshold in normal and STZ-induced diabetic rats treated with or without PKCbeta-selective inhibitor LY333531 (LY) and by assessing the implication of LY in either nNOS-cGMP system or TTX-R Na channels of isolated dorsal root ganglion neurons. The decreased nociceptive threshold in diabetic rats was improved either after 4 weeks of LY treatment or with a single intradermal injection into the footpads. The treatment of LY for 6 weeks significantly decreased p-PKCbeta and ameliorated a decrease in cGMP content in dorsal root ganglia of diabetic rats. The latter effect was confirmed in ex vivo condition. The treatment with NO donor for 4 weeks also normalized both diabetic hyperalgesia and decreased cGMP content in dorsal root ganglions. The expressions of nNOS and TTX-R Na channels were not changed with LY treatment. These results suggest that LY is effective for treating diabetic hyperalgesia through ameliorating the decrease in the nNOS-cGMP system.