Abstract Introduction: Tobacco use intervention in healthcare settings is consistent with best practice guidelines and has been shown to facilitate tobacco cessation. Such intervention is particularly important in rural and medically underserved areas (MUAs), where tobacco use is elevated, as is cancer mortality. Providers’ knowledge and attitudes about the danger of tobacco use and their role in mitigating the same may play a role in whether their patients receive guideline-compliant care. This study investigates knowledge and attitudes toward tobacco use and cessation care delivery among providers at substance use treatment centers (SUTCs) and medical healthcare centers (MHCs; e.g., Federally Qualified Health Centers) in rural and/or MUAs of Texas to identify modifiable barriers to effective tobacco cessation treatment. Methods: Providers from healthcare centers (12 SUTCs, 9 MHCs) participating in a tobacco-free workplace program responded to a baseline solicitation assessing, among other things, their interest in receiving tobacco cessation care training and resources. In total, 347 providers from SUTCs (n=174) and MHCs (n=173) serving 49.6% of Texas’ counties responded to face-valid items assessing their knowledge and attitudes about tobacco use and provision of tobacco cessation care. Generalized or linear mixed models were used to analyze differences between SUTCs and MHCs. Results: More SUTC than MHC providers accurately identified that adults with mental health and/or non-nicotine substance misuse disorders are 2x more likely to smoke cigarettes than the general population (69% vs. 57%, p<0.008) and that quitting smoking can improve associated mental health symptomatology/substance misuse (61% vs. 54%, p<0.05), but also that patients’ tobacco product use is a personal decision that does not concern the direct service provider (30% vs. 19%, p<0.03). Conversely, more MHC than SUTC providers agreed that smoking cessation counseling is an important part of their job (66% vs. 41%, p<0.001), that their patients who smoke want to quit (38% vs. 21%, p<0.002), that providers should advise patients to quit even if it is not the reason for the visit (65% vs. 43%, p<0.001), that counseling by a provider motivates tobacco product users to quit (68% vs. 50%, p<0.005), and that providers should make appointments to specifically help patients quit tobacco product use (59% vs. 39%, p<0.003). Overall, only 48.4% of providers felt somewhat or completely prepared to help patients quit (56% MHC vs. 41% SUTCs, p<0.03). Conclusion: Addressing tobacco use in rural and/or MUAs of Texas where patients receive healthcare is critical to cancer prevention and achieving health equity. Responding SUTC and MHC providers evinced a need for training to enhance their understanding of tobacco use and to elevate the importance of cessation care provision. This is particularly the case in SUTCs, where providers primarily treat addiction. Findings will inform workplace interventions and their adaptation for use in each setting to facilitate guideline-compliant care provision. Citation Format: Sriya N. Kakarla, Brian J. Carter, Ammar D. Siddiqi, Maggie Britton, Tzuan A. Chen, Isabel Martinez Leal, Teresa Williams, Kathleen Casey, Hector Sanchez, Lorraine R. Reitzel. Comparing baseline knowledge and attitudes towards tobacco use and cessation among providers at substance use treatment centers and medical healthcare centers in rural and/or medically underserved areas of Texas [abstract]. In: Proceedings of the 17th AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2024 Sep 21-24; Los Angeles, CA. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2024;33(9 Suppl):Abstract nr A090.