Background: Previous studies suggest that when combined with exercise, Aureobasidium pullulans-derived β-glucan (APβG) may enhance muscle health and fitness profiles because of its ability to improve exercise-induced fatigue and preserve muscle mass. Objectives: The objective was to examine the combined effects and safety of APβG consumption and regular resistance exercise for 12 weeks on muscle strength, biomarkers, and fitness profiles in adults with relatively low skeletal muscle mass. Methods: We conducted a randomized, double-blinded, placebo-controlled trial in adults aged ≥50 years with <110% of the standard lean mass. Eighty participants randomly received either 1000 mg of APβG per day or a placebo for 12 weeks. All participants underwent resistance training three times per week. At baseline and 12 weeks after treatment, we assessed their knee extension/flexion strength, handgrip strength, body composition, and biomarkers. We also evaluated Euro-QoL-5D (EQ-5D) questionnaire, food intake, and physical activity at baseline and 12 weeks after treatment. Results: The combination of APβG and regular resistance exercise over 12 weeks resulted in a higher right knee flexion strength by 4.49 Nm (95% CI; -0.12-8.86 Nm; P = 0.044) than the placebo according to the intention-to-treat analysis. The combination intervention also led to a higher right knee flexion strength of 5.60 Nm (0.18-11.02 Nm; P = 0.043) and left knee flexion strength of 7.25 Nm (0.22-14.28 Nm; P = 0.043) than the placebo according to the per-protocol (PP) analysis. In addition, compared to the placebo, the combined intervention enhanced right-hand grip strength by 1.40 kg (0.19-2.61 kg; P = 0.024) and left-hand grip strength by 1.33 kg (0.01-2.65 kg; P = 0.048) according to PP analysis. The combined intervention also resulted in a more significant reduction in the time required for the 400 m walk test than the placebo. None of the participants experienced adverse events. Conclusion: APβG, in addition to regular resistance exercise, may enhance skeletal muscle strength and fitness in adults with relatively low skeletal muscle mass.