ImportancePrice transparency regulations aim to help patients make informed decisions about medical care, but enforcing these rules is a policy challenge. There may be an association between financial penalties and hospital compliance for enforcing price transparency regulations.ObjectiveTo evaluate the association between financial penalties and acute care hospital compliance with the 2021 Centers for Medicare & Medicaid Services (CMS) Price Transparency Rule.Design, Setting, and ParticipantsThis cohort study uses an instrumental variable design to evaluate the responses of 4377 acute care hospitals in the US operating in 2021 and 2022 to changes in financial penalties in the context of a federal rule requiring hospitals to disclose privately negotiated prices.ExposureChanges in noncompliance penalties between 2021 and 2022 based on a nonlinear function of bed counts.Main Outcomes and MeasuresWhether hospitals publicly posted a machine-readable file with private, payer-specific negotiated prices at the service-code level. Negative controls were used to address confounding.ResultsThe final sample included 4377 hospitals. Compliance increased from 70.4% (n = 3082) in 2021 to 87.7% (n = 3841) in 2022, with 90.2% of hospitals (n = 3948) reporting prices in at least 1 year. Noncompliance penalties increased from $109 500/y in 2021 to a mean (SD) of $510 976 ($534 149)/y in 2022. Penalties in 2022 were substantial, averaging 0.49% of total hospital revenue, 0.53% of total hospital costs, and 1.3% of total employee wages. Compliance increases were significantly positively correlated with penalty increases: a $500 000 increase in penalty was associated with a 2.9–percentage point (95% CI, 1.7-4.2 percentage points; P < .001) increase in compliance. Results were robust to controlling for observable hospital characteristics. No associations were found for preperiod (2021) compliance or ranges of bed counts where penalties do not vary.Conclusions and RelevanceIn this cohort study of 4377 hospitals, compliance with the CMS Price Transparency Rule was associated with increased financial penalties. These findings are relevant for the enforcement of other regulations designed to promote transparency in health care.