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Caffeine consumption inhibits acupuncture analgesic effects by blocking adenosine signaling. However, existing evidence remains controversial. Hence, this study aimed to examine adenosine A1 receptor (A1R) role in moderate-dose caffeine-induced abolishing effect on acupuncture analgesia using A1R knockout mice (A1R). We assessed the role of A1R in physiological sensory perception and its interaction with caffeine by measuring mechanical and thermal pain thresholds and administering A1R and A2AR antagonists in wild-type (WT) and A1R mice. Formalin- and complete Freund’s adjuvant (CFA)-induced inflammatory pain models were recruited to explore moderate-dose caffeine effect on pain perception and acupuncture analgesia in WT and A1R mice. Moreover, a C-fiber reflex electromyogram in the biceps femoris was conducted to validate the role of A1R in the caffeine-induced blockade of acupuncture analgesia. We found that A1R was dispensable for physiological sensory perception and formalin- and CFA-induced hypersensitivity. However, genetic deletion of A1R impaired the antinociceptive effect of acupuncture in A1R mice under physiological or inflammatory pain conditions. Acute moderate-dose caffeine administration induced mechanical and thermal hyperalgesia under physiological conditions but not in formalin- and CFA-induced inflammatory pain. Moreover, caffeine significantly inhibited EA analgesia in physiological and inflammatory pain in WT mice, comparable to that of A1R antagonists. Conversely, A1R deletion impaired the EA analgesic effect and decreased the caffeine-induced inhibitory effect on EA analgesia in physiological conditions and inflammatory pain. Moderate-dose caffeine administration diminished EA-induced antinociceptive effect by blocking A1R. Overall, our study suggested that caffeine consumption should be avoided during acupuncture treatment. PERSPECTIVE: Moderate-dose caffeine injection attenuated EA-induced antinociceptive effect in formalin- and CFA-induced inflammatory pain mice models by blocking A1R. This highlights the importance of monitoring caffeine intake during acupuncture treatment.