Glutathione (GSH) plays a central role in maintenance of cellular redox homeostasis and protection against oxidative injury. Ultraviolet B (UV-B) irradiation–induced GSH depletion is believed to be involved in the pathogenesis of several cutaneous disorders. In this study, the molecular mechanism(s) of UV-B–induced GSH depletion was investigated in cultured human keratinocytes, HaCaT cells. We found that UV-B irradiation caused GSH depletion in a dose- and time-dependent manner in HaCaT cells. The mechanistic studies showed that UV-B–induced GSH depletion did not result from the GSH efflux. UV-B irradiation appeared to cause a slight decrease in enzymatic activity of γ-glutamate cysteine ligase (GCL), a rate-limiting enzyme in GSH biosynthesis. UV-B irradiation resulted in the GCL cleavage through the activation of a caspase cascade. Inhibition of total caspase activity by the general caspase inhibitor, zVAD-fmk, partially reversed the UV-B–induced GSH depletion. More importantly, we found that UV-B irradiation could dramatically decrease the cystine uptake through the functional inhibition of the system Xc⁻, a cystine transporter on the cell membrane. The results suggest that the inactivation of cystine transporter system Xc⁻ was a major contributor to the UV-B–mediated decrease of GSH levels in human keratinocytes.