Background: Appendicular skeletal muscle (or lean) mass (ALM) estimated using dual-energy X-ray absorptiometry (DXA) is considered to be a preferred method for sarcopenia studies. However, DXA is expensive, has limited portability, and requires radiation exposure. Bioelectrical impedance analysis (BIA) is inexpensive, easy to use, and portable; thus BIA might be useful in sarcopenia investigations. However, a large variety of models have been commercially supplied by different companies, and for most consumer products, the equations estimating ALM are not disclosed. It is therefore difficult to use these equations for research purposes. In particular, the BIA equation is often age-dependent, which leads to fundamental difficulty in examining age-related ALM loss. The aims of the current study were as follows: (1) to develop and validate an equation to estimate ALM using multi-frequency BIA (MF-BIA) based on theoretical models, and (2) to establish sarcopenia cutoff values using the equation for the Japanese population. Methods: We measured height (Ht), weight, and ALM obtained using DXA and a standing-posture 8-electrode MF-BIA (5, 50, 250 kHz) in 756 Japanese individuals aged 18 to 86-years-old (222 men and 301 women as developing equation group and 97 men and 136 women as a cross validation group). The traditional impedance index (Ht2/Z50) and impedance ratio of high and low frequency (Z250/Z5) of hand to foot values were calculated. Multiple regression analyses were conducted with ALM as dependent variable in men and women separately. Results: We created the following equations: ALM = (0.6947 × (Ht2/Z50)) + (−55.24 × (Z250/Z5)) + (−10,940 × (1/Z50)) + 51.33 for men, and ALM = (0.6144 × (Ht2/Z50)) + (−36.61 × (Z250/Z5)) + (−9332 × (1/Z50)) + 37.91 for women. Additionally, we conducted measurements in 1624 men and 1368 women aged 18 to 40 years to establish sarcopenia cutoff values in the Japanese population. The mean values minus 2 standard deviations of the skeletal muscle mass index (ALM/Ht2) in these participants were 6.8 and 5.7 kg/m2 in men and women, respectively. Conclusion: The current study established and validated a theoretical and age-independent equation using MF-BIA to estimate ALM and provided reasonable sarcopenia cutoff values.