The antiviral and immunomodulatory role of vitamin D has been shown in various viral infections. However, there is scanty literature available about the effect of vitamin D supplementation in herpes simplex virus-1 (HSV-1) infection. Therefore, the present study aimed to evaluate the role of two different forms of vitamin D: 25-hydroxyvitamin D3 (25D3) and 1,25-dihydroxyvitamin D3 (1,25D3) against HSV-1 in HeLa cells. The HeLa cells were supplemented with either 25D3 or 1,25D3 before HSV-1 infection and were studied after 6, 12, and 24 h postinfection (p.i.). The mRNA levels of toll-like receptors (TLRs), (2, 3, 4, 7, and 9), vitamin D signaling genes, and HSV-1 were studied using real-time PCR. The HSV-1 DNA load was estimated in culture supernatant. The supplementation of 25D3 and 1,25D3 significantly downregulated the mRNA levels of TLR2 (p < 0.0001) at 12 h p.i. The mRNA levels of TLR9 were found to be significantly downregulated in 1,25D3-supplemented cells at 12 h p.i. Furthermore, the significant downregulation was observed in HSV-1 titer in both 25D3- and 1,25D3-supplemented cells at 24 h p.i.(p < 0.0001). However, the effect of 25D3 supplementation persisted till 24 h p.i. with significant downregulation of TLR2 (p < 0.05) mRNA levels. The supplementation of both 25D3 and 1,25D3 before HSV-1 infection was found to downregulate the viral titer and TLR2 mRNA during the intial phase of infection. However, the effect of 25D3 supplementation was found to last for a longer duration compared with 1,25D3.