Natural cis-antisense siRNAs (cis-nat-siRNAs) are a recently characterized class of small regulatory RNAs that are widespread in eukaryotes. Despite their abundance, the importance of their regulatory activity is largely unknown. The only functional role for eukaryotic cis-nat-siRNAs that has been described to date is in environmental stress responses in plants. Here we demonstrate that cis-nat-siRNA-based regulation plays key roles in Arabidopsis reproductive function, as it facilitates gametophyte formation and double fertilization, a developmental process of enormous agricultural value. We show that male gametophytic kokopelli (kpl) mutants display frequent single-fertilization events, and that KPL and a inversely transcribed gene, ARIADNE14 (ARI14), which encodes a putative ubiquitin E3 ligase, generate a sperm-specific nat-siRNA pair. In the absence of KPL, ARI14 RNA levels in sperm are increased and fertilization is impaired. Furthermore, ARI14 transcripts accumulate in several siRNA biogenesis pathway mutants, and overexpression of ARI14 in sperm phenocopies the reduced seed set of the kokopelli mutants. These results extend the regulatory capacity of cis-nat-siRNAs to development by identifying a role for cis-nat-siRNAs in controlling sperm function during double fertilization. -nat-siRNAs (Okamura and Lai 2008;Werner et al. 2009). In plants, there are two studies demonstrating regulatory roles of cis-nat-siRNAs generated from two coding RNAs. In each case, one transcript of the cis-natsiRNA pair was constitutively expressed and the other was induced by stress, either abiotic (Borsani et al. 2005) or biotic (Katiyar-Agarwal et al. 2006). In both cases, down-regulation of the target gene by specific cis-natsiRNAs conferred tolerance to the inductive stress.In flowering plants, the male gametophyte (pollen grain) contains three cells: a vegetative cell that forms