Importance Evidence comparing the consequences of Choosing Wisely recommendations across health systems or with the consequences of recommendations plus policy change is lacking. Objectives To compare changes in the use of 2 low-value laboratory tests after the release of Choosing Wisely recommendations across 3 health care jurisdictions and changes associated with a related policy change. Design, Setting, and Participants This cross-sectional study was a population-based interrupted time series of adult patients (aged 18-64 years) who had primary care visits between January 1, 2010, and June 30, 2015, or established hypothyroidism between January 1, 2012, and June 30, 2015, across 3 health care delivery jurisdictions: Ontario, Canada; the US Veterans Health Administration; and the US employer-sponsored insurance market. Data analysis was performed from March 21, 2018, to October 31, 2019. Exposures A December 2010 payment policy change that eliminated reimbursement of vitamin D screening in Ontario, Canada, and the subsequent release of Choosing Wisely recommendations against low-value use of vitamin D tests in February 2013 and triiodothyronine tests in October 2013 in the United States and both tests in October 2014 in Canada. Main Outcomes and Measures Relative marginal effects (RMEs) comparing low-value testing rates after the release of Choosing Wisely recommendations with rates expected based on prerelease trends and the associated change in low-value vitamin D testing after the 2010 payment policy change in Ontario, Canada. Results Of 54 223 448 total persons, 28 504 576 (52.6%) were female, with 17 895 458 persons (33.0%) aged 18 to 34 years, 11 101 985 (20.5%) aged 35 to 44 years, and 25 226 005 (46.5%) aged 45 to 64 years. The December 2010 policy eliminating reimbursement for low-value vitamin D screening in Ontario, Canada, was associated with a 92.7% (95% CI, 92.4%-93.0%) relative reduction in such screening. Corresponding Choosing Wisely recommendations were associated with smaller reductions: 4.5% (95% CI, 2.6%-6.3%) in Ontario, 13.8% (95% CI, 11.8%-15.9%) for US Veterans Health Administration, and 14.0% (95% CI, 12.8%-15.2%) for US employer-sponsored insurance. In contrast, low-value use of triiodothyronine testing did not change significantly in Ontario, Canada (RME, 0.3%; 95% CI, -1.4% to 2.0%) or the US Veterans Health Administration (RME, 0.7%; 95% CI, -4.7% to 6.4%) and increased (RME, 3.0%; 95% CI, 1.6%-4.4%) for US employer-sponsored insurance. Conclusions and Relevance In this study, marginal reductions in the use of 2 low-value laboratory tests were associated with the release of related Choosing Wisely recommendations but a greater reduction in low-value vitamin D screening was associated with a previous payment policy change implemented in Ontario, Canada. These findings suggest that recommendations alone may be insufficient to significantly reduce use of low-value services and that pairing recommendations with policy changes may be more effective. This cross-sectional study uses rates of vitamin D and triiodothyronine level testing to examine the association of recent policies, recommendations, and systems of care with changes in the use of low-value services at regional and national levels in Canada and the United States. Question Did use of low-value blood tests for vitamin D and triiodothyronine levels change after implementation of a payment policy change or Choosing Wisely recommendations in Canada or the United States? Finding In this cross-sectional study of administrative claims data, a greater reduction in low-value vitamin D screening was associated with a payment policy change and related recommendations in Ontario, Canada, compared with Choosing Wisely recommendations in both Canada and the United States. Reductions in low-value triiodothyronine level testing after relevant recommendations were not observed. Meaning The findings suggest that recommendations alone may be insufficient for reducing use of low-value services at a national or regional level.