LY2605541 is a novel basal insulin analog with a prolonged duration of action. Two Phase I studies assessed LY2605541 pharmacokinetics (PK), glucodynamics (GD), and tolerability in healthy subjects. In Study 1, 33 subjects received single subcutaneous (SC) doses of LY2605541 (0.01–2.22 U/kg) and insulin glargine (0.5–0.8 U/kg) followed by euglycemic clamp for up to 24–36 hours. In Study 2, absolute bioavailability of SC LY2605541 was assessed in 8 subjects by comparing dose normalized area under concentration versus time curve of SC against IV administration. Time-to-maximum plasma concentration (medians) and geometric means for half-life (t1/2) and apparent clearance, respectively, ranged from 18.0 to 42.0 hours, 24.4–45.5 hours, and 1.8–2.8 L/h for SC LY2605541, versus 10.0–12.0 hours, 12.2–14.9 hours, and 51.4–65.2 L/h for SC insulin glargine. LY2605541 glucose infusion rate (GIR) profiles were sustained for ≥36 hours versus glargine GIR profiles, which waned at 24 hours. After IV administration, LY2605541's geometric mean t1/2 was 2.3 hours. LY2605541 intra-subject variability (CV%) was <18% for PK and <32% for GD parameters. The most common adverse events were related to study procedures and were mild-moderate in severity. These results established a well-tolerated baseline dose for LY2605541 with a relatively flat PK profile and low intra-subject variability.