Background Mycobacterium abscessus (Mabs), a rapidly growing Mycobacterium species, is considered an MDR organism. Among the standard antimicrobial multi-drug regimens against Mabs, amikacin is considered as one of the most effective. Parenteral amikacin, as a consequence of its inability to penetrate inside the cells, is only active against extracellular mycobacteria. The use of inhaled liposomal amikacin may yield improved intracellular efficacy by targeting Mabs inside the cells, while reducing its systemic toxicity. Objectives To evaluate the colocalization of an amikacin liposomal inhalation suspension (ALIS) with intracellular Mabs, and then to measure its intracellular anti-Mabs activity. Methods We evaluated the colocalization of ALIS with Mabs in eukaryotic cells such as macrophages (THP-1 and J774.2) or pulmonary epithelial cells (BCi-NS1.1 and MucilAir), using a fluorescent ALIS and GFP-expressing Mabs, to test whether ALIS reaches intracellular Mabs. We then evaluated the intracellular anti-Mabs activity of ALIS inside macrophages using cfu and/or luminescence. Results Using confocal microscopy, we demonstrated fluorescent ALIS and GFP-Mabs colocalization in macrophages and epithelial cells. We also showed that ALIS was active against intracellular Mabs at a concentration of 32 to 64 mg/L, at 3 and 5 days post-infection. Finally, ALIS intracellular activity was confirmed when tested against 53 clinical Mabs isolates, showing intracellular growth reduction for nearly 80% of the isolates. Conclusions Our experiments demonstrate the intracellular localization and intracellular contact between Mabs and ALIS, and antibacterial activity against intracellular Mabs, showing promise for its future use for Mabs pulmonary infections.