Background <p>Laser acupuncture (LA) represents a promising non-invasive neuromodulation technique, yet its therapeutic potential and neurophysiological mechanisms remain largely unexplored compared to traditional needle acupuncture.</p> Objective <p>To investigate the effects of LA at different motor-related acupoints on corticospinal excitability (CSE) and behavioural performance.</p> Methods <p>A single-blinded, randomized, sham-controlled crossover study was conducted with 18 healthy adults. Each participant completed four sessions (LA at LI4, LI10, LI11, and sham) in randomized order. Primary outcomes included cortical excitability measures via transcranial magnetic stimulation. Secondary outcomes assessed behavioural performance using Purdue Pegboard test and Choice Reaction Time tasks via PsyToolkit. The impact of participant-related factors on outcomes was evaluated.</p> Results <p>All active LA conditions significantly improved CSE compared to pre-intervention (<i>p</i> &lt; 0.01), with reduced intracortical inhibition and increased facilitation. LI4 and LI10 demonstrated superior CSE modulation versus sham (p &lt; 0.01), while LI11 showed no significant difference. LI4 additionally improved reaction time performance (p &lt; 0.05), and LI10 exhibited significant immediate post-intervention effects within the session. Higher participant expectancy correlated with greater CSE enhancement (p &lt; 0.05).</p> Conclusions <p>LA at LI4 and LI10 could enhance CSE and behavioural performance, potentially through GABAergic and glutamatergic pathways, as suggested by changes in intracortical excitability. These findings support LA as a non-invasive neuromodulation technique and highlight the importance of acupoint specificity and expectancy effects in cortical excitability outcomes.</p> Clinical trials registration <p>Australian New Zealand Clinical Trials Registry: ACTRN12625000337471.</p>