Hydrogen sulfide (H₂S) has been focused as a regulator of physiological functions and an endogenous antioxidant. A pathway to generate H₂S from D-cysteine has been identified in 2013. D-amino acid oxidase (DAO) is involved in this pathway and especially abundant in the cerebellum among the various brain regions. We have recently demonstrated that D-cysteine prevents the progression of motor dysfunction in a mouse model of spinocerebellar ataxia (SCA). In addition, chaperone-mediated autophagy (CMA) and microautophagy (mA), lysosomal protein degradation pathways, are impaired by the overexpression of several SCA causing proteins. Therefore, we assume that the therapeutic effect of D-cysteine is mediated by the activation of CMA and/or mA through the generation of H₂S. In the present study, we investigated the effects of D-cysteine and a H₂S donor (Na₂S) on CMA/mA activity. We used the GAPDH-HT, a fusion protein of GAPDH with HaloTag (HT), as a marker of CMA/mA activity. In AD293 cells, Na₂S significantly activated CMA/mA, while D-cysteine did not affect the activity because of the lack of DAO expression in AD293 cells. In contrast, CMA/mA was activated by both D-cysteine and Na₂S in primary culture cerebellar Purkinje cells. These findings suggest that D-cysteine activates CMA/mA through the generation of H₂S.