Background and Purpose. Dry needling is an effective therapy for the treatment of pain associated with myofascial trigger point (MTrP). However, the biochemical effects of dry needling that are associated with pain, inflammation, and hypoxia are unclear. This study investigated the activities of β‐endorphin, substance P, TNF‐α, COX‐2, HIF‐1α, iNOS, and VEGF after different dosages of dry needling at the myofascial trigger spots (MTrSs) of a skeletal muscle in rabbit. Materials and Methods. Dry needling was performed either with one dosage (1D) or five dosages (5D) into the biceps femoris with MTrSs in New Zealand rabbits. Biceps femoris, serum, and dorsal root ganglion (DRG) were sampled immediately and 5 d after dry needling for β‐endorphin, substance P, TNF‐α, COX‐2, HIF‐1α, iNOS, and VEGF immunoassays. Results. The 1D treatment enhanced the β‐endorphin levels in the biceps femoris and serum and reduced substance P in the biceps femoris and DRG. The 5D treatment reversed these effects and was accompanied by increase of TNF‐α, COX‐2, HIF‐1α, iNOS, and VEGF production in the biceps femoris. Moreover, the higher levels of these biochemicals were still maintained 5 d after treatment. Conclusion. Dry needling at the MTrSs modulates various biochemicals associated with pain, inflammation, and hypoxia in a dose‐dependent manner.