Background Maternal morbidity and mortality is still a major public health issue in low- and middle-income countries such as Cambodia. Improving access to antenatal care (ANC) services for pregnant women has been widely recognized as one of the most effective means of reducing maternal mortality and morbidity. As such, this study examined the barriers for pregnant women living in rural, agricultural villages to accessing ANC based on data collected in the Ratanakiri province, one of the least developed provinces in Cambodia, using a combination of a community-based cross-sectional survey and a geographic information system (GIS). Methods A community-based cross-sectional survey was conducted among 377 mothers with children under the age of two living in 62 villages in the Ratanakiri province, Cambodia, in December 2015. Face-to-face interviews were conducted to ask mothers about their ANC service use, knowledge of ANC, barriers to accessing health facilities, and complications they experienced during the most recent pregnancy. At the same time, GIS data were also collected using a Global Positioning System (GPS) to accurately measure actual travel distance of pregnant women to access health facilities and to examine geographical and environmental barriers in greater detail. Results Only a third of the mothers met the recommendations made by the World Health Organization (WHO) of receiving ANC four times or more (achieved ANC4+), and a quarter of the mothers had never received ANC during their most recent pregnancy. Factors positively associated with achieving ANC4+ were mother’s secondary or higher education (adjusted odds ratio [AOR] = 5.50, 95% confidence interval [CI]: 1.74, 17.37), being aware that receiving ANC is recommended (AOR = 2.74, 95% CI: 1.25, 6.00), and knowledge about the recommended frequency for ANC (AOR = 2.26, 95% CI: 7.22). Actual travel distance was negatively associated with achieving ANC4+. Mothers who had to travel 10.0–14.9 km were 68% less likely (AOR = 0.32, 95% CI: 0.10, 0.99), and those who had to travel 15.0 km or longer were 79% less likely (AOR = 0.21, 95% CI: 0.07, 0.62) to have achieved ANC 4+, both compared to those who travelled 5.0 km or less. While most previous studies have used a straight-line to measure distance traveled, this study much more accurately measured the actual distance traveled by using a GIS. As a result, there was a statistically significant discrepancy between actual travel distance and straight-line distance. Conclusions This study revealed promoting factors and barriers for ANC use among pregnant women living in remote, agricultural villages in Cambodia. Furthermore, this study highlights the importance of measuring travel distances accurately to ensure that targeted interventions for ANC are not misguided by straight-line distances. The methodology used in this study can be applied widely to other developing countries, especially in remote areas with limited road networks where there may be a large discrepancy between actual and straight-line distances.