Background: Cannabidiol (CBD) has demonstrated anti-inflammatory, analgesic, anxiolytic and neuroprotective effects that have the potential to benefit athletes. This pilot study investigated the effects of acute, oral CBD treatment on physiological and psychological responses to aerobic exercise to determine its practical utility within the sporting context. Methods: On two occasions, nine endurance-trained males (mean ± SD V̇O2max: 57.4 ± 4.0 mL·min−1·kg−1) ran for 60 min at a fixed intensity (70% V̇O2max) (RUN 1) before completing an incremental run to exhaustion (RUN 2). Participants received CBD (300 mg; oral) or placebo 1.5 h before exercise in a randomised, double-blind design. Respiratory gases (V̇O2), respiratory exchange ratio (RER), heart rate (HR), blood glucose (BG) and lactate (BL) concentrations, and ratings of perceived exertion (RPE) and pleasure–displeasure were measured at three timepoints (T1–3) during RUN 1. V̇O2max, RERmax, HRmax and time to exhaustion (TTE) were recorded during RUN 2. Venous blood was drawn at Baseline, Pre- and Post-RUN 1, Post-RUN 2 and 1 h Post-RUN 2. Data were synthesised using Cohen’s dz effect sizes and 85% confidence intervals (CIs). Effects were considered worthy of further investigation if the 85% CI included ± 0.5 but not zero. Results: CBD appeared to increase V̇O2 (T2: + 38 ± 48 mL·min−1, dz: 0.25–1.35), ratings of pleasure (T1: + 0.7 ± 0.9, dz: 0.22–1.32; T2: + 0.8 ± 1.1, dz: 0.17–1.25) and BL (T2: + 3.3 ± 6.4 mmol·L−1, dz: > 0.00–1.03) during RUN 1 compared to placebo. No differences in HR, RPE, BG or RER were observed between treatments. CBD appeared to increase V̇O2max (+ 119 ± 206 mL·min−1, dz: 0.06–1.10) and RERmax (+ 0.04 ± 0.05 dz: 0.24–1.34) during RUN 2 compared to placebo. No differences in TTE or HRmax were observed between treatments. Exercise increased serum interleukin (IL)-6, IL-1β, tumour necrosis factor-α, lipopolysaccharide and myoglobin concentrations (i.e. Baseline vs. Post-RUN 1, Post-RUN 2 and/or 1-h Post-RUN 2, p’s < 0.05). However, the changes were small, making it difficult to reliably evaluate the effect of CBD, where an effect appeared to be present. Plasma concentrations of the endogenous cannabinoid, anandamide (AEA), increased Post-RUN 1 and Post-RUN 2, relative to Baseline and Pre-RUN 1 (p’s < 0.05). CBD appeared to reduce AEA concentrations Post-RUN 2, compared to placebo (− 0.95 ± 0.64 pmol·mL−1, dz: − 2.19, − 0.79). Conclusion: CBD appears to alter some key physiological and psychological responses to aerobic exercise without impairing performance. Larger studies are required to confirm and better understand these preliminary findings. Trial Registration This investigation was approved by the Sydney Local Health District’s Human Research Ethics Committee (2020/ETH00226) and registered with the Australia and New Zealand Clinical Trials Registry (ACTRN12620000941965).