Single motorcycle accidents are common in Nagano Prefecture where is mountainous areas in Japan. In a previous study, analysis of traffic accident statistics data suggested that the fatality and serious injury rates for uphill right curves and downhill left curves are high, however the true causes of these accidents remain unclear. In this study, a motorcycle simulator was used to evaluate the driving characteristics due to these road alignments. Evaluation courses based on combinations of uphill/downhill slopes and left/right curves were created, and experiments were conducted. The subjects of the study were expert riders and novice riders. The results showed that right curves are even more difficult to see near the entrance of the curve when accompanied by an uphill slope, making it easier to delay recognition and judgment of the curve. Expert riders recognized curves faster than novice riders. Additionally, expert riders take a large lean of the vehicle body, actively attempted to ride on the inside corner, while that of novice riders was less. On the other hand, for downhill left curves, there was a tendency for delayed judgment of sharp curves, and riders were more likely to increase their speed. Also, the expert riders recognized curve curvature earlier and had a greater lean angle of the vehicle body than the novice riders, but there was no significant difference. From these results, the road alignment combination of uphill/downhill slopes and left/right curve has a significant impact on the risk of motorcycle accidents.