Artificial Insemination, the deposition of semen into the reproductive tract of female animal is being increasingly adopted in livestock (cattle and buffalo) breeding in Nepal. A study was carried out at 3 different dairy co-operatives of Mangalpur Village Development Committee (VDC), Chitwan, Nepal during Jan-April, 2015 to assess the knowledge, attitude and practice regarding Artificial Insemination (AI) among smallholder livestock farmers. A questionnaire survey was employed to interview 81 respondents raising 148 livestock animals including 98 cattle and 50 buffaloes. Although 80.25% of the total respondents have known about AI, the practice of AI was found to be low (25.95%) of which a vast majority (95%) was applying in cattle and only 5% in case of buffalo. It was found that the success of conception at first time was in 65.55% of the artificially inseminated animals while 13.72% of them conceived only after repetition of AI. 45.5% of the total respondents did not prefer AI because of varied reasons like lower conception rate, increase in cost due to repetition, unavailability of technicians, easy availability of bull, incidence of reproductive disorders in dam and calf, and possibility of weak calf birth. Meanwhile, 40.74% preferred AI to get progenies with higher yields and better traits, have higher conception rate, minimize the transmission of disease from breeding bull, and for feasibility and economic reasons. It was found that 27.16% of the respondents seemed to adopt AI if the availability of technicians, increased rate of conception, and increased chance of she calf and reasonable cost of insemination could be assured. If these aspects could be addressed appropriately with proper extension activities, AI would be one of the best approaches to uplift the productivity of indigenous dairy livestock animals.