Eukaryotic Argonaute proteins play primary roles in miRNA and siRNA pathways that are essential for numerous developmental and biological processes. However, the functional roles of the four ZmAGO1 genes have not yet been characterized in maize (Zea mays L.). In the present study, ZmAGO1a was identified from four putative ZmAGO1 genes for further characterization. Complementation of the Arabidopsis ago1-27 mutant with ZmAGO1a indicated that constitutive overexpression of ZmAGO1a could restore the smaller rosette, serrated leaves, later flowering and maturation, lower seed set, and darker green leaves at late stages of the mutant to the wild-type phenotype. The expression profiles of ZmAGO1a under five different abiotic stresses indicated that ZmAGO1a shares expression patterns similar to those of Argonaute genes in rice, Arabidopsis, and wheat. Further, variation in ZmAGO1a alleles among diverse maize germplasm that resulted in several amino acid changes revealed genetic diversity at this locus. The present data suggest that ZmAGO1a might be an important AGO1 ortholog in maize. The results presented provide further insight into the function of ZmAGO1a.