The hard-shell mussels Mytilus coruscus have been extensively employed in pollution biomonitoring. Earlier studies indicated that metal concentrations in Mytilus coruscus may not accurately reflect the true metal contamination levels in the sampling areas, possibly due to their modified metal uptake and efflux. Given the likelihood of mussels in the field being exposed to intermittent metal contaminants, this study investigated whether different Cu pre-exposures significantly affected its uptake and efflux upon Cu exposure. We found significant reduction in Cu uptake rate constant (ku) and efflux rate constant (ke) in the mussels with varying Cu pre-exposure regimes. Specifically, the ku decreased from 1.55 ± 0.37 L g−1 d−1 in the control group to 0.65 ± 0.19 after 5 days and 0.53 ± 0.28 after 15 days of exposure to 20 μg L−1 Cu, respectively, and then was further reduced to as low as 0.096 ± 0.046 L g−1 d−1 following a 5-day exposure at 50 μg L−1 Cu. Similarly, the ke decreased from 0.18 ± 0.020 to 0.15 ± 0.015 d−1 following 5–15 days of exposure to 20 μg L−1 Cu, and further decreased to 0.081 ± 0.023 d−1 after a 5-day exposure at 50 μg L−1 Cu. Our subcellular distribution analysis underscored the critical role of the metallothionein-like protein (MTLP) fraction in modifying both Cu ku and ke during the rapid-depuration phase (ke1), whereas the metal-rich granule (MRG) fraction influenced the ke during the second depuration phase (ke2). This study demonstrated that environmental assessments utilizing biomonitoring species should consider the exposure of these organisms to ensure accurate interpretations of metal contamination in marine ecosystems and enhance the effectiveness of these species in environmental monitoring. This crucial factor is often overlooked, potentially skewing data and leading to misinterpretations of environmental health and pollution levels.