Studies have explored how vitamin B12 status affects sleep among elders and children, but this remains to be investigated among young adults. We used the Pittsburgh Sleep Quality Index (PSQI) to assess the association between serum vitamin B12 and sleep among female college students in Saudi Arabia. In this cross-sectional study, we enrolled 355 participants (age (years), 20.7 ± 1.5; body mass index, 23.6 kg/m2 ± 5.2) at King Saud University, Riyadh, Saudi Arabia. Fasting blood samples were analyzed regarding the serum vitamin B12 and blood lipids. Anthropometric, socio-demographic, clinical history, stress, physical activity, and dietary data were collected. We assessed the sleep statuses of the participants using the PSQI. Around 72% of the participants were “poor” sleepers (PSQI > 5). Subgroup analysis within the tertiles showed that participants with higher vitamin B12 in the second and third tertiles reported better scores for sleep quality (B ± SE = −12.7 ± 5.6, p = 0.03; B ± SE = −32.7 ± 16.4, p = 0.05, respectively) and also reported a lower use of sleep medication (B ± SE = −21.2 ± 9.9, p = 0.03, in the second tertile only), after adjusting for the waist–hip ratio and stress. However, sleep was not found to be directly associated with either serum vitamin B12 or dietary vitamin B12. In conclusion, the serum vitamin B12 results show that the participants with higher vitamin B12 in the second and third tertiles reported better scores on the sleep quality scale and a lower use of sleep medication. However, no such associations were observed with the overall PSQI. More studies with larger sample sizes are needed to establish a direct relationship between sleep and vitamin B12.