Objectives: The purpose of this study was to compare the effects of deep dry needling (DN) with and without needle manipulation on pressure pain thresholds (PPTs) and electromyographic (EMG) amplitude of the lumbosacral multifidus (LM) in adults with low back pain (LBP). Methods: Participants were randomized into two treatment groups: with needle manipulation (n = 21) and without needle manipulation (n = 21). All participants received a single session of the assigned DN intervention. PPTs and EMG amplitude of the LM muscle were collected three times: before DN, immediately after DN, and one week after DN. Results: The needle manipulation group had a significantly greater increase in PPT immediately after the intervention and at the one-week follow-up as compared to the no needle manipulation group. The increase of PPT in the needle manipulation group was significant immediately after the intervention, and the increase remained significant at the one-week follow-up. However, there was no significant difference in EMG amplitude of the LM muscle between groups across the three time points. Discussion: Deep DN with needle manipulation appeared to reduce mechanical pressure sensitivity more than DN without manipulation for patients with LBP. Although a single session of DN could reduce pressure pain sensitivity, it may not be sufficient to improve LM muscle function. Level of Evidence: 1b. Trial registration numbers: NCT03970486.