Cannabis is the most commonly used illicit drug in the world. Cannabinoids have been shown to modulate immune responses; however, the association of cannabis with neuroimmune function has never been investigated in vivo in the human brain. To investigate neuroimmune activation or 18-kDa translocator protein (TSPO) levels in long-term cannabis users, and to evaluate the association of brain TSPO levels with behavioral measures and inflammatory blood biomarkers. This cross-sectional study based in Toronto, Ontario, recruited individuals from January 1, 2015, to October 30, 2018. Participants included long-term cannabis users (n = 24) and non-cannabis-using controls (n = 27). Cannabis users were included if they had a positive urine drug screen for only cannabis and if they used cannabis at least 4 times per week for the past 12 months and/or met the criteria for cannabis use disorder. All participants underwent a positron emission tomography scan with 18FFEPPA, or fluorine F 18-labeled N-(2-(2-fluoroethoxy)benzyl)-N-(4-phenoxypyridin-3-yl)acetamide. Total distribution volume was quantified across regions of interest. Stress and anxiety as well as peripheral measures of inflammatory cytokines and C-reactive protein levels were also measured. In total, 24 long-term cannabis users (mean SD age, 23.1 3.8 years; 15 men 63%) and 27 non-cannabis-using controls (mean SD age, 23.6 4.2 years; 18 women 67%) were included and completed all study procedures. Compared with the controls, cannabis users had higher 18FFEPPA total distribution volume (main group effect: F1,48 = 6.5 P = .01; ROI effect: F1,200 = 28.4 P < .001; Cohen d = 0.6; 23.3% higher), with a more prominent implication for the cannabis use disorder subgroup (n = 15; main group effect: F1,39 = 8.5 P = .006; ROI effect: F1,164 = 19.3 P < .001; Cohen d = 0.8; 31.5% higher). Greater TSPO levels in the brain were associated with stress and anxiety and with higher circulating C-reactive protein levels in cannabis users. The results of this study suggest that TSPO levels in cannabis users, particularly in those with cannabis use disorder, are higher than those in non-cannabis-using controls. The findings emphasize the need for more complementary preclinical systems for a better understanding of the role of cannabinoids and TSPO in neuroimmune signaling.