Nitrogen isotope compositions (δ15N values) of kerogen in the sedimentary rocks from the 1878 Ma Gunflint Formation were analysed to understand the relationships among microbial activities, ocean chemistry, and tectonic evolution in the Animikie Basin. In the present study, the stepwise combustion analysis, performed on 13 kerogen samples, indicates that the δ15N values of kerogen can be sub-divided into two fractions with discrete values depending on the combustion temperatures: a lower-temperature fraction (from 500 to 575 °C), and a higher-temperature fraction (higher than 575 °C). A positive correlation was observed between the δ15N values of the lower-temperature fractions and Pr/Sm ratios, which represent contributions from the continental input. In contrast, no correlation was observed between the δ15N values of the higher-temperature fractions and the Pr/Sm ratios. This relationship between the δ15N values and the continental inputs is explained by the isotopic fractionation effects of the biological nitrogen cycle associated with the enhanced microbial activity, triggered by the active tectonic settings in the Animikie basin.