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Introduction Pregnancy is a natural process that may create some changes in different parts of the body including the oral cavity. These changes will lead to oral diseases if enough and timely care of oral cavity is not taken. Women may experience increased gingivitis or pregnancy gingivitis beginning in the second or third month of pregnancy that increases in severity throughout the duration of pregnancy. To motivate the patient toward oral health and implement the needed prophylactic measures, a longitudinal study was planned to observe the effect of oral health education during pregnancy on knowledge, attitude, practice, oral health status, and treatment needs (TNs) of pregnant women belonging to different socioeconomic groups. Materials and methods A longitudinal study was conducted among 112 pregnant women belonging to different socioeconomic groups to assess the effect of oral health education on knowledge, attitude, practice, oral health status, and TNs. The demographic details, knowledge, attitude, and practice of pregnant women, and oral health status were collected through a predesigned questionnaire by a principal investigator through an interview. Oral health examination was carried out to assess oral health status using revised World Health Organization Proforma 1997, and oral health education was given through PowerPoint presentation to the participants in local language, i.e., Marathi, after collecting the baseline data. Reinforcement of oral health education and blanket referral was done at 14th week, and follow-up data were collected at 28th week of gestation. The demographic details, such as age, sex, education, occupation, income, and the questions based on knowledge, attitude, and practice among participants were analyzed using number, percentage, and mean. Results At baseline, knowledge was limited, attitude was positive, while the practice was poor regarding oral health care during pregnancy in pregnant women belonging to different socioeconomic groups. After oral health education and blanket referral, at 28th week of gestation, knowledge regarding oral health care improved drastically, attitude toward oral health became more positive, whereas practice did not change much among all the pregnant women belonging to different socioeconomic groups, probably indicating sociocultural influences. Conclusion Intensive oral health education during pregnancy leads to drastic improvement in knowledge and attitude. Practice, gingival health, and the number of filled teeth also improved to some extent. Clinical significance Regular oral health education programs should be conducted at community level among pregnant woman to reduce the burden of oral diseases. How to cite this article Chawla RM, Shetiya SH, Agarwal DR, Mitra P, Bomble NA, Narayana DS. Knowledge, Attitude, and Practice of Pregnant Women regarding Oral Health Status and Treatment Needs following Oral Health Education in Pune District of Maharashtra: A Longitudinal Hospital-based Study. J Contemp Dent Pract 2017;18(5):371-377.