INTRODUCTION: Estrogen is protective against weight gain via mechanisms mediated by adipocyte-specific receptors. Specifically, estrogen receptor beta (ERb) activation is non-feminizing and increases adipocyte mitochondrial activity leading to increased energy expenditure (EE). Prior observations that activation of beta 3 adrenergic receptor (b3R) via CL316,243 (CL) increases ERb expression led to the hypothesis that CL may increase ERb ligand sensitivity due to the increased expression of ERb. METHODS: The novel ERb ligand OSU-ERb12 (OSU) and CL were tested both independently and in combination in obese C57/bl mice (n=8-10/group, age at start of treatment: 24 weeks). Following treatments, mice were assessed for body weight (BW), body composition via EchoMRI, energy expenditure (EE) and physical activity (PA) via Prometheon metabolic chamber system (Sable Systems), and glucose tolerance via glucose tolerance testing (GTT). Statistical differences were determined via ANOVA followed by post-hoc Tukey's test to determine between group differences. P-value<0.05 was considered statistically significant. Statistical analyses was performed using SPSS v. 29. RESULTS: CL+OSU significantly reduced BW [CL+OSU v CTRL: p-value=0.004; CL+OSU v OSU: p-value=0.021] and body fat percentage (BF%)[CL+OSU v CTRL: p-value<0.001; CL+OSU v OSU: p-value=0.016] compared to control (CTRL) and OSU despite no change in energy intake [p-value=0.131]. Additionally, CL alone reduced BF% compared to CTRL [p-value=0.037]. There was no change in total lean mass (LM) from pre- to post-treatment [p-value=0.409]. CL+OSU resulted in a significant increase in EE compared to both CTRL and OSU [CL+OSU v CTRL: p-value=0.005; CL+OSU v OSU: p-value<0.001]. While there was a difference in PA between CL and OSU [CL v OSU: p-value=0.010], there was no difference in PA between CL+OSU and any other treatment group [p-values>0.05]. CL and CL+OSU led to significant reductions in fasting blood glucose (BG) and GTT AUC when compared to the groups that did not receive CL: OSU and CTRL [CL+OSU v CTRL, CL+OSU v OSU, CL v CTRL, CL v OSU: p-values<0.001]. DISCUSSION: Findings indicate that CL+OSU induced weight loss via increased EE. The lack of LM reduction despite weight loss indicates a LM preservation effect of CL+OSU. There was no significant difference in PA between OSU+CL and any other group indicating that the increases in EE were driven by increased basal metabolism rather than increased PA or decreased food intake. CL, both with and without OSU, improved glucose tolerance and fasting BG, whereas OSU alone did not. CONCLUSION: The therapeutic combination of CL+OSU had the greatest effect on BF%, suggesting a synergistic effect of CL and OSU on fat loss. However, effects on EE and glucose tolerance were comparable between CL and CL+OSU, indicating CL as a primary driver of these responses. While there was no effect of OSU alone on glucose tolerance, it did not impair the ability of CL to improve glucose tolerance in the combination group. CAFNR CAAT and JOD (internal funding to Dr. Vieira-Potter) This abstract was presented at the American Physiology Summit 2025 and is only available in HTML format. There is no downloadable file or PDF version. The Physiology editorial board was not involved in the peer review process.