Stat3 is initially dephosphorylated in murine keratinocytes in response to UVB irradiation. Treatment with Na(3)VO(4) desensitized keratinocytes to UVB-induced apoptosis with the recovery of phosphorylated Stat3 protein levels, implying that a protein tyrosine phosphatase (PTP) is involved in this mechanism. In the current work, we report that three PTPs including TC45 (the nuclear form of TC-PTP), SHP1, and SHP2 are involved in this rapid dephosphorylation of Stat3 in keratinocytes induced by UVB irradiation. Dephosphorylation of Stat3 was increased rapidly after UVB irradiation of cultured keratinocytes. Knockdown of TC-PTP, SHP1, or SHP2 using RNAi showed that these PTPs are likely responsible for most of the rapid Stat3 dephosphorylation observed following UVB irradiation. The level of phosphorylated Stat3 was significantly higher in keratinocytes transfected with TC-PTP, SHP1, or SHP2 siRNA in the presence or absence of UVB compared with keratinocytes transfected with control siRNA. TC45 was mainly localized in the cytoplasm of keratinocytes and translocated from cytoplasm to nucleus upon UVB irradiation. Stat3 dephosphorylation was associated with nuclear translocation of TC45. Further studies revealed that knockdown of all three phosphatases, using RNAi, prevented the rapid dephosphorylation of Stat3 following UVB irradiation. In mouse epidermis, the level of phosphorylated Stat3 was initially decreased, followed by a significant increase at later time points after UVB exposure. The levels of Stat3 target genes, such as cyclin D1 and c-Myc, followed the changes in activated Stat3 in response to UVB irradiation. Collectively, these results suggest that three phosphatases, TC45, SHP1, and SHP2, are primarily responsible for UVB-mediated Stat3 dephosphorylation and may serve as part of an initial protective mechanism against UV skin carcinogenesis.