Cannabis is among the most used illicit substances in the world, and it is estimated that around 10% of regular cannabis users are susceptible to developing cannabis use disorder (CUD). Our study aimed to clarify the effect of delta-9-tetrahydrocannabinol (THC) on epigenetic DNA modifiers conducting DNA methylation and demethylation (DNMTs and TET enzymes) on human peripheral mononuclear cells (PBMCs) in vitro. Additionally, we profiled the genome-wide effect of THC and assessed if any genes with marked changes in methylation or hydroxymethylation could be identified. Changes in DNA methylation have been implicated in impairing various processes both in the periphery and in the brain and therefore play a role in long-term behavioural changes associated with addiction. Our first repeated THC treatments study revealed that the cells adapted to THC. After the initial changes in DNMTs and TET enzymes mRNA levels and enzymatic activity, at the end of the study period, normalization to the same level as the control group occurred. In our second THC challenge experiment, we saw that repeated THC exposure had sensitized the cells to a THC challenge exposure after a period of abstinence. On a genome-wide level, we saw an increase in hydroxymethylation level after THC challenge treatment. Genes that exhibited the strongest associations with THC exposure had functional significance in signal transmission and cell adhesion. Our results show that THC impacts DNA methylation and demthylation in human PBMCs in vitro. Knowing the epigenetic effects of THC helps us understand the complex processes underlying the development of CUD.