ABSTRACT ACTIN DEPOLYMERIZING FACTORs (ADFs) regulate the organization and dynamics of actin microfilaments (AFs) by cleavage and depolymerization of AFs. The Arabidopsis thaliana genome encodes 11 ADF genes grouped into four subclasses. Subclass I ADF s, ADF1 , -2 , -3 , and -4 , are expressed in all vegetative tissues and are reportedly involved in the regulation of plant growth, and biotic and abiotic stress responses. Furthermore, the nuclear localization of ADF4 is seemingly important in disease responses. Here, we present data that indicate a previously unknown regulatory role of subclass I ADFs in leaf senescence. ADF4 knockout mutants ( adf4 ) and transgenic lines in which the expression of all members of subclass I ADF s was downregulated ( ADF1-4Ri ) showed acceleration of both dark-induced and age-dependent leaf senescence. Among the eight ACTIN genes encoded in A. thaliana , ACT2 , -7 , and -8 are expressed in vegetative tissues. In contrast to adf4 and ADF1-4Ri , neither ACT2 and ACT8 double mutant ( act2/8 ), nor ACT7 knockout mutant ( act7 ), showed accelerated leaf senescence. Upregulation of the expression of senescence-associated genes occurred earlier in adf4 and ADF1-4Ri lines than in wild type. Examination of the expression of subclass I ADF s genes during senescence revealed a reduced expression of ADF4 but not of other subclass I members. Additionally, we showed that nuclear localization of ADF4 was important for regulating leaf senescence. Altogether, our data indicate that subclass I ADFs, particularly ADF4, play an important role in the regulation of leaf senescence.