Direct manipulations of neuronal activity have been shown to induce changes in DNA methylation (DNAm), although little is known about the cellular signalling pathways involved. Using reduced representation bisulfite sequencing we identify DNAm changes associated with moderate chronic depolarization in dissociated rat hippocampal cultures. Consistent with previous findings, these changes occurred primarily in the vicinity of loci implicated in neuronal function, being enriched in intergenic regions and under-represented in CpG-rich promoter regulatory regions. We subsequently used two pharmacological interventions (nifedipine and FK-506) to test whether the identified changes depended on two inter-related signalling pathways known to mediate multiple forms of neuronal plasticity. Both pharmacological manipulations had notable effects on the extent and magnitude of depolarization-induced DNAm changes indicating that a high proportion of activity-induced changes are likely to be mediated by calcium entry through L-type CaV1 channels and/or downstream signalling via the calcium-dependent phosphatase calcineurin.