Background: Stable isotope analysis is being utilized with increasing regularity to examine a wide range of issues (diet, habitat use, migration) in ecology, geology, archaeology, and related disciplines. A crucial component to these studies is a thorough understanding of the range and causes of baseline isotopic variation, which is relatively poorly understood for nitrogen (d 15 N). Animal excrement is known to impact plant d 15 N values, but the effects of seabird guano have not been systematically studied from an agricultural or horticultural standpoint.Methodology/Principal Findings: This paper presents isotopic (d 13 C and d 15 N) and vital data for maize (Zea mays) fertilized with Peruvian seabird guano under controlled conditions. The level of 15 N enrichment in fertilized plants is very large, with d 15 N values ranging between 25.5 and 44.7% depending on the tissue and amount of fertilizer applied; comparatively, control plant d 15 N values ranged between 20.3 and 5.7%. Intraplant and temporal variability in d 15 N values were large, particularly for the guano-fertilized plants, which can be attributed to changes in the availability of guano-derived N over time, and the reliance of stored vs. absorbed N. Plant d 13 C values were not significantly impacted by guano fertilization. High concentrations of seabird guano inhibited maize germination and maize growth. Moreover, high levels of seabird guano greatly impacted the N metabolism of the plants, resulting in significantly higher tissue N content, particularly in the stalk. Conclusions/Significance:The results presented in this study demonstrate the very large impact of seabird guano on maize d 15 N values. The use of seabird guano as a fertilizer can thus be traced using stable isotope analysis in food chemistry applications (certification of organic inputs). Furthermore, the fertilization of maize with seabird guano creates an isotopic signature very similar to a high-trophic level marine resource, which must be considered when interpreting isotopic data from archaeological material.