The aim of this exploratory study was to investigate how sedentary behavior (SB) and physical activity (PA) influence DNA methylation at a global, gene-specific, and health-related pathway level. SB, light PA (LPA), and moderate-to-vigorous PA (MVPA) were assessed objectively for 41 Flemish men using the SenseWear Pro 3 Armband. CpG site-specific methylation in leukocytes was determined using the Illumina HumanMethylation 450 BeadChip. Correlations were calculated between time spent on the three PA intensity levels and global DNA methylation, using a z-score-based method to determine global DNA methylation levels. To determine whether CpG site-specific methylation can be predicted by these three PA intensity levels, linear regression analyses were performed. Based on the significantly associated CpG sites at α = 0.005, lists were created including all genes with a promoter region overlapping these CpG sites. A biological pathway analysis determined to what extent these genes are overrepresented within several pathways. No significant associations were observed between global DNA methylation and SB (r = 0.084), LPA (r = -0.168), or MVPA (r = -0.125), although the direction of the correlation coefficients is opposite to what is generally reported in literature. SB has a different impact on global and gene-specific methylation than PA, but also LPA and MVPA affect separate genes and pathways. Furthermore, the function of a pathway seems to determine its association with SB, LPA, or MVPA. Multiple PA intensity levels, including SB, should be taken into account in future studies investigating the effect of physical (in)activity on human health through epigenetic mechanisms.