Background: Effective and timely treatment of vitamin B12 deficiency in childhood is crucial because it can lead to serious issues, including delayed growth and neuromotor development. Available treatment options include oral, intramuscular, and sublingual administration. Aim: This study investigated the efficacy of a new dosing protocol for sublingual methylcobalamin. Materials and Methods: In total, 312 patients with vitamin B12 deficiency (serum level < 250 ng/L) were retrospectively enrolled between September 2022 and April 2023. The patients were divided into two groups based on their treatment type: intramuscular cyanocobalamin (Group 1) and sublingual methylcobalamin (Group 2). Group 1 included 29 (9.3%) patients and Group 2 included 283 (90.7%) patients, with 56 (18%) patients in Group 2 undergoing treatment for childhood cancer. The sublingual methylcobalamin protocol consisted of 1 puff (500 μg) daily for children under 8 years of age and 2 puffs (1000 μg) daily for those 8 years and older, administered for 1.5 months and then three times weekly for an additional 1.5 months. The efficacy of this protocol was compared to the standard intramuscular cyanocobalamin regimen. Results: The mean ages in Groups 1 and 2 were 10.07 ± 6.05 years (range, 1–17 years) and 7.43 ± 5.86 years (range, 0.1–17 years), respectively. The female/male ratio was 19/10 in Group 1 and 145/138 in Group 2. The most common diagnoses were anaemia (72, 22.9%), cancer (56, 18.0%), and haemangioma (40, 12.7%). The median serum levels of vitamin B12 in Group 1 were 177 ng/L before treatment, 447 ng/L after 1.5 months, and 321.5 ng/L after 3 months. In Group 2, the levels were 172 ng/L before treatment, 438 ng/L after 1.5 months, and 360 ng/L after 3 months. There were no significant between-group differences. Both groups showed a statistically significant increase in levels above 300 ng/L. In Group 2, the haemoglobin and red blood cell levels showed statistically significant changes, while the mean corpuscular volume, white blood cell count, and platelet count did not substantially change in either group. Conclusion: Sublingual methylcobalamin, a noninvasive treatment option, was as effective as intramuscular cyanocobalamin. This study is the first to compare the standard intramuscular protocol with a new dosing regimen for sublingual methylcobalamin. Sublingual methylcobalamin should be considered an effective alternative, particularly for patients with absorption issues that make oral vitamin B12 insufficient and for those in whom intramuscular administration carries a risk of muscle and sciatic nerve damage.