Introduction: Trans and gender-diverse (TGD) individuals often face stigma and discrimination in healthcare, hindering access to gender-affirming care. Training healthcare workers on TGD health aims to foster inclusive and affirming care practices. This review aimed to evaluate the effectiveness of TGD health training programs for healthcare workers. Methods: This systematic review followed the PRISMA guidelines and was registered with PROSPERO (CRD42023443288). We searched 13 databases for studies up to March 2024, with no language/geographic restrictions. Ten reviewers screened studies in pairs, resolving discrepancies via discussion or third-reviewer input. We included randomized/non-randomized comparative and before-after studies for quantitative analysis (mean difference [MD] or standardized mean difference [SMD] with 95% CIs) and qualitative/mixed-methods studies for thematic synthesis. Evidence certainty was assessed using GRADE (quantitative) and GRADE-CERQual (qualitative). Outcomes included knowledge, attitudes, skills, discrimination, competence, comfort, TGD quality of life, and stakeholder preferences. Results: From 20,188 records, 85 studies were included. Training appears to have improved healthcare workers' knowledge (SMD=1.08, 95% CI 0.78-1.39), attitudes (SMD=0.22, 95% CI 0.05-0.39), skills (SMD=0.96, 95% CI 0.56-1.37), competence (SMD=0.55, 95% CI 0.29-0.81), and comfort (SMD=0.69, 95% CI 0.17-1.21). Qualitative analysis of 130 findings identified 18 categories and four key themes on intervention design and impact. Conclusions: TGD training programs may enhance health workers' knowledge, attitudes, skills, competence, and comfort. Well-structured, interactive, and inclusive programs showed promise, but evidence certainty was low with limited follow-up. Further high-quality research is needed to confirm these findings.