Background: Depot formulations prolong the analgesic effect of local anesthetics and reduce peak plasma drug concentration. This allows for safer administration of larger doses of local anesthetics, which further prolongs the duration of analgesic effect. We previously reported the development of large multivesicular vesicles (LMVVs) remotely loaded with bupivacaine (LMVV liposomal bupivacaine) and demonstrated a >5-fold prolongation of analgesic effect in animals and humans. In this study, we present pharmacokinetic data of LMVV liposomal bupivacaine in humans. Methods: Healthy volunteers received subcutaneous injections of 20 mL plain 0.5% bupivacaine and, 1 week later, 20 mL of 2% LMVV liposomal bupivacaine in a prospective, open-label, crossover, controlled study. Results: Eight subjects were studied. No subjective side effects of local anesthetics were observed. The maximal plasma concentration and the time to achieve maximal plasma concentration were assessed by modeling plasma concentration-time profiles. Maximal plasma concentration was not significantly different between groups (0.87 ± 0.45 μg/mL and 0.83 ± 0.34 μg/mL for plain and liposomal bupivacaine, respectively; P = not significant, 0.83). These values are well below the putative toxic plasma concentration of 2 to 4 μg/mL. Time to achieve maximal plasma concentration was 7-fold greater for the liposomal preparation (262 ± 149 minutes vs 37.5 ± 16 minutes, P < 0.01). Conclusions: Peak plasma bupivacaine concentrations were not different in the 2 groups, despite a 4-fold increase in total bupivacaine dose administered in the novel liposomal preparation. The delayed elimination and prolonged redistribution of liposomal bupivacaine to plasma is compatible with the depot-related slow-release effect leading to the prolonged pharmacodynamic effect previously reported.