Technical Abstract: Pollen tubes navigate through different female tissues and deliver the sperm to the embryo sac for fertilization. Protein disulfide isomerases play important roles in the maturation of secreted or plasma membrane proteins. Here we show that truncated versions of a protein disulfide isomerase (PDI), PDIL2-1, cause a delay in embryo sac maturation and thereby disrupt pollen tube guidance in Arabidopsis. Plants carrying either of two T-DNA insertions in the coding region of PDIL2-1 had truncated transcripts and a reduced seed set phenotype. Reciprocal crosses indicate that these mutations acted sporophytically, and aniline blue staining and scanning electron microscopy showed that funicular and micropylar pollen tube guidance were disrupted. A PDIL2-1-YFP fusion protein was mainly localized in the ER and was expressed in all tissues examined, but in ovules, expression was detected in integument tissues, with much higher levels in the micropylar region in later developmental stages, but was never detected in embryo sacs. We show that PDIL2-1 is a functional enzyme. Expression of another copy of the full-length protein or of truncated versions that retained enzyme activity resulted in reduced seed set, but expression of a version in which enzyme activity was abolished did not, indicating that the T-DNA insertion lines are gain-of-function mutants. Our results suggest that these versions of PDIL2-1 function in sporophytic tissues to affect ovule structure and impede embryo sac development, thereby disrupting pollen tube guidance.