IMPORTANCE: 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. OBJECTIVE: 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. DESIGN, SETTING, AND PARTICIPANTS: 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. MAIN OUTCOMES AND MEASURES: 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. RESULTS: 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. CONCLUSIONS AND RELEVANCE: 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.