Low and middle-income countries have extended state sponsored Social Health Insurance (SHI) to people outside the formal sector to enhance access to healthcare. However, in spite of the relatively low costs of signing up and the benefits offered by SHIs, up-take rates are very low among the informal sector populations. The objective of this study was to investigate factors affecting participation of people in the informal sector in the National Hospital Insurance Fund (NHIF) scheme in Kakamega County, Kenya. This cross-sectional study was conducted in Kakamega County in Western Kenya. The study employed a mixed methods designs approach. In the first phase of the study, 400 participants were recruited using both probability and non-probability sampling methods; 400 persons engaged in informal sector activities were recruited through random sampling. In the second phase of the study, 24 key informants and 5 groups consisting of 8-12 persons were purposively selected for in-depth interviews and Focus Group Discussions respectively. The study established that people in the informal sector with higher income (> Kshs.10, 000) are more likely to enroll (odds ratio 2.21 with 95% CI: 1.07 to 4.03) compared to those with low incomes and similarly, higher level of education was significantly associated with enrolment in NHIF scheme (odds ratio 31.07 with 95% CI: 17.19 to 87.94). Rigid scheme design features create difficulties for people in informal sector to participate. In conclusion, policy decisions should focus on interventions directed at educating poor populations, people with low educational levels and those working outside formal employments on the benefits of subscribing to the NHIF scheme in Kenya. The study recommends subsidies or waivers to increase affordability of participation in the NHIF scheme, particularly for people with low monthly incomes.