Objectives: Although well-being at work is important for occupational health, multi-dimensional workplace well-being measures do not exist for Japanese workers. The purpose of this study was to investigate the validity of the Japanese version of the Workplace PERMA-Profiler. Methods: Japanese workers completed online surveys at baseline (N = 310) and 1 month later (N = 100). The Workplace PERMA-Profiler was translated according to international guidelines. Job and life satisfaction, work engagement, psychological distress, work-related psychosocial factors, and work performance were measured as comparisons for convergent validity. Cronbach's alphas, Intra-class Correlation Coefficients (ICCs), and measurement errors were calculated for the reliability, and the validity of the measure was tested by correlational analyses and confirmatory factor analysis. Results: A total of 310 (baseline) and 86 (follow-up) workers responded and were included in the analyses. Cronbach's alphas and ICCs of the Japanese Workplace PERMA-Profiler ranged from 0.75 to 0.96. Confirmatory factor analysis indicated that the 5-factor model demonstrated a marginally acceptable fit (χ2 (80) = 351.30, CFI = 0.892, TLI = 0.858, RMSEA = 0.105, SRMR = 0.051). Overall well-being and the five PERMA domains had moderate-to-strong correlations with job satisfaction, psychological distress (inversely), and work-related factors. Conclusions: The Japanese version of the Workplace PERMA-Profiler demonstrated adequate reliability and validity. This measure could be useful to assess well-being at work, promote well-being research among Japanese workers, and address the problem of definition for well-being in further studies.