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Background: Cervical cancer is one of the most common cancers among women worldwide. The incidence rate in Indian women between 25 to 44 years is about 3.5%, and age standardised incidence rate is 22 per 1,00,000 women. In rural areas, it is a major health problem, where the disease is diagnosed usually at a late stage. Cervical cancer has a long window for diagnosis, during which the precancerous lesions can be diagnosed by various screening methods. Pap smear is one such method. It is cost effective and can be done easily in an outpatient set-up. For satisfactory implementation of the screening programme for early diagnosis of pre-cancerous lesions, the services of the nursing staff can be utilised to educate and motivate all eligible women of the community to take Pap smear. The present study was conducted to assess the knowledge about cervical cancer and Pap smear practice among the nursing staff of the rural hospital in Maharashtra, India. Materials and Method: This is a cross-sectional descriptive study performed during 1st December, 2020 and 28th February, 2021. A total of 320 staff nurses participated in the study. A structured questionnaire was given to each of them after obtaining verbal consent. The answered questionnaires were collected for further analysis. Results: Majority of the participants were between 21 and 35 years of age. 91.56% of the participants were married, 97.19% (N=311) were aware about cervical cancer, while 88.75% (N=284) were aware about Pap smear. Among the 311 respondents, 35.05% knew that Human papillomavirus is a causative agent of cervical cancer, and 33.75% are aware about HPV vaccination. Even though 47.19% of the 284 participants were trained to take Pap smear, 48.51% of those trained had not taken any Pap smear. Among the married group, 37.06% (N= 293) had undergone Pap smear screening themselves. Conclusion: Though the knowledge about cervical cancer and Pap smear is adequate among the participants, the practice is not satisfactory. The nursing staff need to be encouraged, educated and trained to practice the screening method for themselves and for other women of the community so that the morbidity and mortality from cervical cancer is controlled. [ABSTRACT FROM AUTHOR]