MRNA vaccines have been shown to provide strong immune responses against SARS-CoV-2, but the extent of their antibody cross-reactivity against human seasonal coronaviruses, such as NL63, remains unclear. Furthermore, it is unknown whether pre-existing antibody responses against NL63 might affect the outcome of SARS-CoV-2 mRNA vaccination. This study used a flow cytometry-based serological assay and an in vitro neutralization assay to analyze NL63 antibody responses in sera from SARS-CoV-2 mRNA-vaccinated mice and plasma samples from a vaccinated human cohort. We found that the Moderna mRNA-1273 vaccine can generate cross-reactive antibodies against NL63. Importantly, SARS-CoV-2 mRNA vaccination did not boost pre-existing anti-NL63 responses in humans, and pre-existing NL63 antibody levels did not affect the antibody response induced by SARS-CoV-2 mRNA vaccination. These findings suggest that while SARS-CoV-2 mRNA vaccination can induce cross-reactive antibodies against NL63, pre-existing immunity to this seasonal coronavirus does not appear to significantly impact the effectiveness of the vaccine. This contributes to our understanding of the complex interplay between pre-existing immunity to seasonal coronaviruses and the immune response generated by SARS-CoV-2 mRNA vaccines.