Objective: Shoulder exoskeletons can effectively assist with overhead work. However, their impacts on muscle synergy remain unclear. The objective is to systematically investigate the effects of the shoulder exoskeleton on muscle synergies during overhead work.Methods: Eight male participants were recruited to perform a screwing task both with (Intervention) and without (Normal) the exoskeleton. Eight muscles were monitored and muscle synergies were extracted using non-negative matrix factorization and electromyographic topographic maps. Results: The number of synergies extracted was the same (n = 2) in both conditions. Specifically, the first synergies in both conditions were identical, with the highest weight of AD and MD; while the second synergies were different between conditions, with highest weight of PM and MD, respectively. As for the first synergy in the Intervention condition, the activation profile significantly decreased, and the average recruitment level and activation duration were significantly lower (p<0.05). The regression analysis for the muscle synergies across conditions shows the changes of muscle synergies did not influence the sparseness of muscle synergies (p=0.7341). In the topographic maps, the mean value exhibited a significant decrease (p<0.001) and the entropy significantly increased (p<0.01). Conclusion: The exoskeleton does not alter the number of synergies and existing major synergies but may induce new synergies. It can also significantly decrease neural activation and may influence the heterogeneity of the distribution of monitored muscle activations. Significance: This study provides insights into the potential mechanisms of exoskeleton-assisted overhead work and guidance on improving the performance of exoskeletons.