H2S induces somatic and visceral hyperalgesia via Cav3.2 T-type Ca2+ channels (T-channels) and also TRPA1 channels. Interestingly, ascorbic acid (AA) selectively inhibits Cav3.2, and AA administration inhibits H2S-induced hyperalgesia and neuropathic pain. Here we examined effect of AA deficiency on H2S-evoked somatic or visceral hyperalgesia, using SMP30/GNL knockout (KO) mice that are incapable of synthesizing AA and dependent on dietary AA. After weaning, the KO mice were fed on an AA-deficient diet and had free access to water containing sufficient (1.5 g/L) AA [AA (+)] or only minimum AA (0.0375 g/L) [AA (−)] for seven weeks. The AA content dramatically decreased in the brain, spinal cord, plasma, hindpaw and colon from AA (−) KO mice, but not AA (+) KO mice, although AA deficiency was not induced by dietary control in wild-type (WT) mice. In WT mice, intraplantar (i.pl.) and intracolonic (i.col.) administration of NaHS, an H2S donor, caused hyperalgesia in the hindpaw and lower abdomen, respectively, as assessed by von Frey test, which was blocked by T-channel inhibitors or AA. In AA (−) KO mice, i.pl. and i.col. NaHS produced greater somatic and colonic pronociceptive activities, respectively, as compared with AA (+) KO mice and WT mice. In contrast, AA deficiency did not alter the pronociceptive effects of ally isothiocyanate, a TRPA1 agonist, in KO mice. Together, AA appears to modulate H2S-induced somatic/visceral pain most probably by limiting Cav3.2 function (1489).